ErbB4 antagonists

ABSTRACT

The present invention concerns methods and means for controlling excessive proliferation and/or migration of smooth muscle cells, and in particular for treating stenosis, by using antagonists of a native ErbB4 receptor. The invention further concerns a method for the identification of ErbB4 agonists and antagonists capable of inhibiting or enhancing the proliferation or migration of smooth muscle cells.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.10/362,380, filed Aug. 6, 2003, which is the National Stage Applicationfiled under 35 U.S.C. §371 of PCT/US01/26984, filed Aug. 29, 2001, whichis a continuation of U.S. patent application Ser. No. 09/940,101 filedAug. 27, 2001, now abandoned, which claims benefit under 35 U.S.C. §119(e) of U.S. Provisional Patent Application Ser. No. 60/265,516 filedJan. 31, 2001 and of U.S. Provisional Patent Application Ser. No.60/229,679 filed Sep. 1, 2000, the entire disclosures of which arehereby expressly incorporated by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention concerns methods and means for controllingexcessive proliferation and/or migration of smooth muscle cells, and inparticular for treating stenosis, by using antagonists of a native ErbB4receptor. The invention further concerns a method for the identificationof ErbB4 agonists and antagonists capable of inhibiting or enhancing theproliferation or migration of smooth muscle cells.

2. Description of the Related Art

1. ErbB Receptor Tyrosine Kinases

Transduction of signals that regulate cell growth and differentiation isregulated in part by phosphorylation of various cellular proteins.Protein tyrosine kinases are enzymes that catalyze this process.Receptor protein tyrosine kinases are believed to direct cellular growthvia ligand-stimulated tyrosine phosphorylation of intracellularsubstrates.

HER4/Erb4 is a receptor protein tyrosine kinase belonging to the ErbBfamily. Increased ErbB4 expression closely correlates with certaincarcinomas of epithelial origin, including breast adenocarcinomas(Plowman et al., Proc. Natl. Acad. Sci. USA 90:1746-1750 [1993]; Plowmanet al., Nature 366:473-475 [1993]). Diagnostic methods for detection ofhuman neoplastic conditions (especially breast cancers) which evaluateErbB4 expression are described in EP Pat Appln No. 599,274.

Other members of the ErbB family of receptor tyrosine kinases include:epidermal growth factor receptor (EGFR), ErbB2 (HER2/neu), and ErbB3(HER3). The erbB1 gene encodes the 170 kDa epidermal growth factorreceptor (EGFR) that has been causally implicated in human malignancy.In particular, increased expression of this gene has been observed inmore aggressive carcinomas of the breast, bladder, lung and stomach(Modjtahedi, H. and Dean, C. (1994) Int. J. Oncol. 4:277-296). HER4acts, in the absence of HER2, as a mediator of antiproliferative anddifferentiative response in human breast cancer cell lines. (Sartor etal., Mol. Cell Biol. 21:4265-75 (2001)).

The neu gene (also called erbB2 and HER2) encodes a 185 kDa receptorprotein tyrosine kinase that was originally identified as the product ofthe transforming gene from neuroblastomas of chemically treated rats.Amplification and/or overexpression of the human HER2 gene correlateswith a poor prognosis in breast and ovarian cancers (Slamon, D. J. etal., Science 235:177-182 (1987); Slamon et al., Science 244:707-712(1989); and U.S. Pat. No. 4,968,603). Overexpression of HER2 (frequentlybut not uniformly due to gene amplification) has also been observed inother carcinomas including carcinomas of the stomach, endometrium,salivary gland, lung, kidney, colon, thyroid, pancreas and bladder.

A further related gene, called erbB3 or HER3, has been described. SeeU.S. Pat. No. 5,183,884; Kraus et al., Proc. Natl. Acad. Sci. USA86:9193-9197 (1989); EP Pat Appln No 444,961A1; and Kraus et al., Proc.Natl. Acad. Sci. USA 90:2900-2904 (1993). Kraus et al. (1989) discoveredthat markedly elevated levels of erbB3 mRNA were present in certainhuman mammary tumor cell lines indicating that erbB3, like erbB1 anderbB2, may play a role in human malignancies. They also showed thatEGF-dependent activation of the ErbB3 catalytic domain of a chimericEGFR/ErbB3 receptor resulted in a proliferative response in transfectedNIH-3T3 cells. Furthermore, these researchers demonstrated that somehuman mammary tumor cell lines display a significant elevation ofsteady-state ErbB3 tyrosine phosphorylation further indicating that thisreceptor may play a role in human malignancies. The role of erbB3 incancer has been explored by others. It has been found to beoverexpressed in breast (Lemoine et al., Br. J. Cancer 66:1116-1121(1992)), gastrointestinal (Poller et al., J. Pathol. 168:275-280 (1992),Rajkumer et al., J. Pathol. 170:271-278 (1993), and Sanidas et al., Int.J. Cancer 54:935-940 (1993)), and pancreatic cancers (Lemoine et al., J.Pathol. 168:269-273 (1992), and Friess et al., Clinical Cancer Research1:1413-1420 (1995)). ErbB3 is unique among the ErbB receptor family inthat it possesses little or no intrinsic tyrosine kinase activity (Guyet al., Proc. Natl. Acad. Sci. USA 91:8132-8136 (1994) and Kim et al. J.Biol. Chem. 269:24747-55 (1994)).

The ErbB receptors are generally found in various combinations in cellsand heterodimerization is thought to increase the diversity of cellularresponses to a variety of ErbB ligands (Earp et al. Breast CancerResearch and Treatment 35: 115-132 (1995)). EGFR is bound by sixdifferent ligands; epidermal growth factor (EGF), transforming growthfactor alpha (TGF-α), amphiregulin, heparin binding epidermal growthfactor (HB-EGF), β-cellulin and epiregulin (Groenen et al. GrowthFactors 11:235-257 (1994)). A family of heregulin proteins resultingfrom alternative splicing of a single gene are ligands for ErbB3 andErbB4. The heregulin family includes α, β and β heregulins (Holmes etal., Science, 256:1205-1210 (1992); U.S. Pat. No. 5,641,869; andSchaefer et al. Oncogene 15:1385-1394 (1997)); neu differentiationfactors (NDFs), glial growth factors (GGFs); acetylcholine receptorinducing activity (ARIA); and sensory and motor neuron derived factor(SMDF). For a review, see Groenen et al. Growth Factors 11:235-257(1994); Lemke, G. Molec. & Cell. Neurosci. 7:247-262 (1996) and Lee etal. Pharm. Rev. 47:51-85 (1995). Recently three additional ErbB ligandswere identified; neuregulin-2 (NRG-2) which is reported to bind eitherErbB3 or ErbB4 (Chang et al. Nature 387 509-512 (1997); and Carraway etal Nature 387:512-516 (1997)); neuregulin-3 which binds ErbB4 (Zhang etal. PNAS (USA) 94(18):9562-7 (1997)); and neuregulin-4 which binds ErbB4(Harari et al. Oncogene 18:2681-89 (1999)). HB-EGF, β-cellulin andepiregulin also bind to ErbB4.

While EGF and TGFα do not bind ErbB2, EGF stimulates EGFR and ErbB2 toform a heterodimer, which activates EGFR and results intransphosphorylation of ErbB2 in the heterodimer. Dimerization and/ortransphosphorylation appear to activate the ErbB2 tyrosine kinase. SeeEarp et al., supra. Likewise, when ErbB3 is co-expressed with ErbB2, anactive signaling complex is formed and antibodies directed against ErbB2are capable of disrupting this complex (Sliwkowski et al., J. Biol.Chem., 269(20):14661-14665 (1994)). Additionally, the affinity of ErbB3for heregulin (HRG) is increased to a higher affinity state whenco-expressed with ErbB2. See also, Levi et al., Journal of Neuroscience15: 1329-1340 (1995); Morrissey et al., Proc. Natl. Acad. Sci. USA 92:1431-1435 (1995); and Lewis et al., Cancer Res., 56:1457-1465 (1996)with respect to the ErbB2-ErbB3 protein complex. ErbB4, like ErbB3,forms an active signaling complex with ErbB2 (Carraway and Cantley, Cell78:5-8 (1994)).

Because of the physiological importance, members of the ErbB family ofreceptor tyrosine kinases have often been targeted for therapeuticdevelopment. For example, Hudziak et al., Mol. Cell. Biol.9(3):1165-1172 (1989) describe the generation of a panel of anti-ErbB2antibodies one of which, called 4D5, inhibited cellular proliferation by56%. A recombinant humanized version of the murine anti-ErbB2 antibody4D5 (huMAb4D5-8, rhuMAb HER2 or HERCEPTIN®; U.S. Pat. No. 5,821,337) isclinically active in patients with ErbB2-overexpressing metastaticbreast cancers that have received extensive prior anti-cancer therapy(Baselga et al., J. Clin. Oncol. 14:737-744 (1996)). HERCEPTIN® receivedmarketing approval from the Food and Drug Administration Sep. 25, 1998for the treatment of patients with metastatic breast cancer whose tumorsoverexpress the ErbB2/HER2 protein. Since HER2 is also overexpressed inother cancers, in addition to breast cancer, HERCEPTIN® holds a greatpotential in the treatment of such other cancers as well.

2. Smooth Muscle Cell Proliferation

Smooth muscle cells are very important structural and functionalcomponents of many hollow passages in the body, including blood vessels,gastrointestinal tract, airway passage (trachea and bronchi in lungs),urinary tract system (bladder and ureters) etc. They are responsible forelasticity that is so crucially required for normal functioning of theseorgans. They respond to a variety of physiological stimuli byconstriction or dilation as needed, for example, for regulating the flowof fluids carried by them. They respond not only to chemical stimuli,such as growth factors and cytokines, but also to physical stimuli, suchas pressure and stretch. Excessive proliferation of smooth muscle cellsresults in thickening of the wall and narrowing the lumen of the organsknown as “stenosis” in a variety of disorders.

A number of growth factors and cytokines are implicated in theproliferation of smooth muscle cells. One category of such importantmolecules are EGF related ligands. For example, smooth muscle cells froma variety of such organs have been demonstrated to possess EGFreceptors, and some of them even synthesize and secrete EGF ligands suchas HB-EGF, thus setting up autocrine loop. Various EGF ligands act aspotent mitogens and stimulate proliferation of smooth muscle cells oftenresulting in thickening of the wall and ultimately stenosis. Forexample, excessive proliferation of vascular smooth muscle cells (VSMC)is involved in pathology of vascular stenosis, restenosis resulting fromangioplasty or surgery or stent implants, atherosclerosis, transplantatherosclerosis and hypertension (reviewed in Casterella and Teirstein,Cardiol. Rev. 7: 219-231 [1999]; Andres, Int. J. Mol. Med. 2: 81-89[1998]; and Rosanio et al., Thromb. Haemost. 82 [suppl 1]: 164-170[1999]). The thickening of blood vessels increases resistance to bloodflow and ultimately leads to hypertension. Moreover, decreased bloodsupply to the tissue may also cause necrosis and induce inflammatoryresponse leading to severe damage. For example, myocardial infarctionoccurs as a result of lack of oxygen and local death of heart muscletissues.

Infantile hypertrophic pyloric stenosis (IHPS), which causes functionalobstruction of the pyloric canal also involves hypertrophy andhyperplasia of the pyloric smooth muscle cells (Oue and Puri, Pediatr.Res. 45: 853-857 [1999]). Furthermore, EGF, EGF receptor and HB-EGF areimplicated in pathogenesis of pyloric stenosis (Shima et al., Pediatr.Res. 47: 201-207 [2000]).

Similarly, the urinary bladder wall thickening that occurs in responseto obstructive syndromes affecting the lower urinary tract involvesproliferation of urinary bladder smooth muscle cells. A membrane-boundprecursor form of HB-EGF is expressed in urinary bladder smooth musclecells and HB-EGF is a potent mitogen for bladder SMC proliferation(Freeman et al., J. Clin. Invest. 99: 1028-1036 [1997]; Kaefer et al.,J. Urol. 163: 580-584 [2000]; Borer et al., Lab Invest. 79: 1335-1345[1999]).

The obstructive airway diseases are yet another group of diseases withunderlying pathology involving smooth muscle cell proliferation. Oneexample of this group is asthma which manifests in airway inflammationand bronchoconstriction. EGF is implicated in the pathologicalproliferation of airway SMCs in obstructive airway diseases (Cerutis etal., Am. J. Physiol. 273: L10-15 [1997]; Cohen et al., Am. J. Respir.Cell. Mol. Biol. 16: 85-90 [1997]).

The instant invention discloses the use of ErbB4 receptor antagonistsfor controlling excessive migration and/or proliferation or smoothmuscle cells and, in particular, for the treatment of stenosis.

SUMMARY OF THE INVENTION

In one aspect, the invention concerns a method for controlling excessiveproliferation or migration of smooth muscle cells by treating the smoothmuscle cells with an effective amount of an antagonist of a native ErbB4receptor. The control is prevention or inhibition, including totalinhibition, of excessive proliferation or migration of smooth musclecells. In one embodiment the smooth muscle cells are urinary bladdersmooth muscle cells, and in another embodiment they are the smoothmuscle cells of an airway passage.

The excessive proliferation or migration of smooth muscle cells such asvascular smooth muscle cells may result in stenosis including vascularstenosis and restenosis. In one embodiment the smooth muscle cells arehuman. The stenosis may be further characterized by excessiveproliferation or migration of endothelial cells.

In one embodiment the ErbB4 receptor antagonist is an immunoadhesin. Inanother embodiment the ErbB4 receptor antagonist is an antibody, such asa neutralizing antibody against a native ErbB4 receptor.

In another aspect, the invention concerns a method for treating stenosisin a mammalian patient, including a human, comprising administering tothe patient an effective amount of an antagonist of a native mammalianErbB4 receptor. The treatment includes prevention of stenosis. Thestenosis may be vascular stenosis including restenosis. The antagonistmay be administered as an injection or infusion. The treatment may alsobe used to reduce hypertension associated with the stenosis. Thestenosis may be vascular stenosis including restenosis, pyloricstenosis, thickening of the urinary bladder wall or part of anobstructive airway disease.

In one embodiment the antagonist is an immunoadhesin, which may comprisethe extracellular region of a native human ErbB4 receptor. In anotherembodiment the antagonist is an antibody, such as a neutralizingantibody against a native human ErbB4 receptor.

In a further aspect, the invention concerns a method for treatingstenosis in a mammalian patient, such as a human, comprising introducinginto a cell of the patient a nucleic acid encoding an antagonist of anErbB4 receptor. The nucleic acid may be introduced in vivo or ex vivo,and with the aid of a vector such as retroviral vector or a lipid-baseddelivery system. The method of the present invention is particularlyuseful for the treatment (including prevention) of vascular stenosis andrestenosis.

The antagonist may be an immunoadhesin. The antagonist may also be anantibody, such as a neutralizing antibody against a native human ErbB4receptor.

In another aspect, the invention concern a method for treatinghypertension associated with vascular stenosis in a mammalian patient,comprising administering to the patient an effective amount of anantagonist of a native ErbB4 receptor. The antagonist may be a smallmolecule.

In a still further aspect, the invention concerns a pharmaceuticalcomposition for the treatment of stenosis in a mammalian patientcomprising an effective amount of an antagonist of a native mammalianErbB4 receptor, in admixture with a pharmaceutically acceptable carrier.

In all aspects, preferred ErbB4 antagonists include immunoadhesins,preferably comprising a native human ErbB4 receptor extracellular domainsequence fused to an immunoglobulin constant region sequence. Theimmunoglobulin sequence preferably is that of a heavy chain constantregion of an IgG1, IgG2 or IgG3 immunoglobulin and may additionallycomprise an immunoglobulin light chain sequence covalently attached tothe fusion molecule comprising the immunoglobulin heavy chain constantregion.

Another preferred class of ErbB4 antagonists comprises neutralizingantibodies specifically binding a native ErbB4 receptor. The antibodiespreferably are human or humanized. In one embodiment the antibodies bindessentially the same epitope as an antibody produced by a hybridomaselected from the group consisting of HER4.10H1.1A1 (ATCC AccessionNumber PTA-2828), HER4.1C6.A11 (ATCC Accession Number PTA-2829),HER4.3B9.2C9 (ATCC Accession Number PTA-2826), HER4.1A6.5B3 (ATCCAccession Number PTA-2827) and HER4.8B1.2H2 (ATCC Accession NumberPTA-2825). The antibodies may also have complementarity determiningregion (CDR) residues from an antibody produced by a hybridoma selectedfrom the group consisting of HER4.10H1.1A1 (ATCC Accession NumberPTA-2828), HER4.1C6.A11 (ATCC Accession Number PTA-2829), HER4.3B9.2C9(ATCC Accession Number PTA-2826), HER4.1A6.5B3 (ATCC Accession NumberPTA-2827) and HER4.8B1.2H2 (ATCC Accession Number PTA-2825).

The smooth muscle cells may, for example, be pyloric or urinary bladdersmooth muscle cells, or smooth muscle cells of an airway passage.Preferably, the smooth muscle cells are vascular smooth muscle cells.

In a still further aspect, the invention concerns a method foridentifying a molecule that inhibits or enhances the proliferation ormigration of smooth muscle cells, comprising the steps of: (a)contacting a polypeptide comprising an amino acid sequence having atleast 85% sequence identity with the amino acid sequence of theextracellular domain of a native ErbB4 receptor and retaining theability to control excessive proliferation or migration of smooth musclecells, with a candidate molecule; and (b) determining whether thecandidate molecule inhibits or enhances the ability of the polypeptideto control excessive proliferation or migration of smooth muscle cells.The polypeptide may comprise the extracellular domain of a native ErbB4receptor. The polypeptide is an immunoadhesin in one embodiment. In aparticular embodiment, the molecule enhances the ability of thepolypeptide to control excessive proliferation or migration of smoothmuscle cells, and is an antibody or a small molecule.

In a yet further aspect the invention concerns an antibody that bindsessentially the same epitope of ErbB4 as an antibody produced by ahybridoma selected from the group consisting of HER4.10H1.1A1 (ATCCAccession Number PTA-2828), HER4.1C6.A11 (ATCC Accession NumberPTA-2829), HER4.3B9.2C9 (ATCC Accession Number PTA-2826), HER4.1A6.5B3(ATCC Accession Number PTA-2827) and HER4.8B1.2H2 (ATCC Accession NumberPTA-2825). In addition to the methods set forth above and throughout thedisclosure, these antibodies are believed to be useful in the treatmentof various cancers, including breast cancer.

In a still further aspect the invention concerns an antibody that hascomplementarity determining region (CDR) residues from an antibodyproduced by a hybridoma selected from the group consisting ofHER4.10H1.1A1 (ATCC Accession Number PTA-2828), HER4.1C6.A11 (ATCCAccession Number PTA-2829), HER4.3B9.2C9 (ATCC Accession NumberPTA-2826), HER4.1A6.5B3 (ATCC Accession Number PTA-2827) andHER4.8B1.2H2 (ATCC Accession Number PTA-2825).

In a further aspect the invention concerns an antibody selected from thegroup consisting of an antibody produced by a hybridoma selected fromthe group consisting of HER4.10H1.1A1 (ATCC Accession Number PTA-2828),HER4.1C6.A11 (ATCC Accession Number PTA-2829), HER4.3B9.2C9 (ATCCAccession Number PTA-2826), HER4.1A6.5B3 (ATCC Accession NumberPTA-2827) and HER4.8B1.2H2 (ATCC Accession Number PTA-2825).

The invention also concerns an antibody that binds essentially the sameepitope of ErbB4 bound by an antibody selected from the group consistingof anti-ErbB4 monoclonal antibodies 4-1440, 4-1460, 4-1473, 4-1492 and4-1464.

Further, the invention concerns an antibody that has complementaritydetermining region (CDR) residues from an antibody selected from thegroup consisting of anti-ErbB4 monoclonal antibodies 4-1440, 4-1460,4-1473, 4-1492 and 4-1464.

The invention also concerns an antibody that binds ErbB4 with highaffinity. This antibody preferably binds to ErbB4 with a Kd of less than100 nM, more preferably with a Kd of less than 50 nM, even morepreferably with a Kd of less than 25 nM and most preferably with a Kdless than 10 nM. In one embodiment this antibody is a human antibody andin another embodiment it is a humanized antibody. In yet anotherembodiment the antibody is an antibody fragment.

The invention further concerns an antibody which is capable of bindingto both ErbB4 and ErbB3. In one embodiment the antibody is capable ofbinding ErbB4 with high affinity and in another embodiment the antibodybinds both ErbB4 and ErbB3 with high affinity.

In another aspect the invention concerns an antibody which binds toErbB4 and reduces heregulin binding thereto. This antibody may bindErbB4 with high affinity.

Finally, the invention concerns an antibody which binds to ErbB4 andreduces heregulin-induced tyrosine phosphorylation thereof. Thisantibody may also bind ErbB4 with high affinity.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A, 1B and 1C show the nucleotide sequence of human ErbB4 (SEQ IDNO: 1).

FIG. 2 shows the deduced amino acid sequence of human ErbB4 (SEQ ID NO:2).

FIGS. 3A and 3B show the nucleotide sequence of an ErbB4-IgGimmunoadhesin (SEQ ID NO: 3).

FIG. 4 shows the amino acid sequence of the ErbB4 extracellular domain(ECD), which comprises amino acids 26 through 640 (SEQ ID NO: 4) of theErbB4 amino acid sequence presented in FIG. 2 (SEQ ID NO: 2).

FIG. 5 shows the effect of ErbB4-IgG immunoadhesin on PDGF-stimulatedproliferation of human aortic smooth muscle cells.

FIG. 6 shows the effect of ErbB4-IgG immunoadhesin on the chemotacticresponse of human aortic smooth muscle cells to thrombin.

FIG. 7 shows the inhibition of heregulin binding to HER4 immunoadhesinby anti-HER4 monoclonal antibodies.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT A. Definitions

Unless defined otherwise, technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. See, e.g. Singleton et al.,Dictionary of Microbiology and Molecular Biology 2nd ed., J. Wiley &Sons (New York, N.Y. 1994); Sambrook et al., Molecular Cloning, ALaboratory Manual, Cold Springs Harbor Press (Cold Springs Harbor, N.Y.1989). For purposes of the present invention, the following terms aredefined below.

Unless indicated otherwise, the term “ErbB” when used herein refers toany one or more of the mammalian ErbB receptors (i.e. ErbB1 or epidermalgrowth factor (EGF) receptor; ErbB2 or HER2 receptor; ErbB3 or HER3receptor; ErbB4 or HER4 receptor; and any other member(s) of this classI tyrosine kinase family to be identified in the future) and “erbB”refers to the mammalian erbB genes encoding these receptors.

The terms “ErbB4” and “HER4” are used interchangeably and refer to anative sequence ErbB4 receptor polypeptide as disclosed, for example, inEuropean Patent Application No. (EP) 599,274; Plowman et al., Proc.Natl. Acad. Sci. USA, 90:1746-1750 (1993); and Plowman et al., Nature,366:473-475 (1993), and functional derivatives, including amino acidsequence variants thereof.

A “native” or “native sequence” ErbB4 or HER4 receptor has the aminoacid sequence of a naturally occurring ErbB4 receptor in any mammalian(including humans) species, irrespective of its mode of preparation.Accordingly, a native or native sequence ErbB4 receptor may be isolatedfrom nature, produced by techniques of recombinant DNA technology,chemically synthesized, or produced by any combinations of these orsimilar methods. Native ErbB4 receptors specifically includepolypeptides having the amino acid sequence of naturally occurringallelic variants, isoforms or spliced variants of ErbB4, known in theart or hereinafter discovered. Native sequence ErbB4 receptors aredisclosed, for example, in EP 599,274, supra, and in the two Plowman etal. papers, supra. Elenius et al., J. Biol. Chem. 272:26761-26768 (1997)report the identification of two alternatively spliced isoforms of ErbB4both in mouse and human tissues, that differ by the insertion of either23 (HER4 JM-a) or 13 (HER4 JM-b) alternative amino acids in theextracellular juxtamembrane (JM) region. Elenius et al., Oncogene18:2607-2615 (1999) report the identification and characterization ofanother naturally occurring isoform of ErbB4 (designated as ErbB4CYT-2), with a deletion of the cytoplasmic domain sequence required forthe activation of the PI3-K intracellular signal transduction pathway.HER4 isoforms are also disclosed in WO 99/19488. A nucleotide sequenceencoding ErbB4 is presented in FIG. 1 (SEQ ID NO: 1) and thecorresponding deduced amino acid sequence is depicted in FIG. 2 (SEQ IDNO: 2).

The term “ErbB4 extracellular domain” or “ErbB4 ECD” refers to a solublefragment of ErbB4 comprising the amino acids located between the signalsequence and the first predicted transmembrane region. In oneembodiment, the “ErbB4 ECD” is a polypeptide comprising amino acids26-640 (SEQ ID NO: 4) of the human ErbB4 sequence presented in FIG. 2(SEQ ID NO: 2).

The term “mammal” is used herein to refer to any animal classified as amammal, including, without limitation, humans, domestic and farmanimals, and zoo, sports, or pet animals, such as sheep, dogs, horses,cats, cows, etc. Preferably, the mammal herein is human.

“Functional derivatives” include amino acid sequence variants, andcovalent derivatives of the native polypeptides as long as they retain aqualitative biological activity of the corresponding native polypeptide.Amino acid sequence variants generally differ from a native sequence inthe substitution, deletion and/or insertion of one or more amino acidsanywhere within a native amino acid sequence. Deletional variantsinclude fragments of the native polypeptides, and variants having N-and/or C-terminal truncations. Ordinarily, amino acid sequence variantswill possess at least about 70% homology, preferably at least about 80%,more preferably at least about 90% homology with a native polypeptide.

“Homology” is defined as the percentage of residues in the amino acidsequence variant that are identical after aligning the sequences andintroducing gaps, if necessary, to achieve the maximum percent homology.Methods and computer programs for the alignment are well known in theart. One such computer program is “Align 2”, authored by Genentech,Inc., which was filed with user documentation in the United StatesCopyright Office, Washington, D.C. 20559, on Dec. 10, 1991.

An ErbB “antagonist” is a molecule, which prevents or interferes with anErbB effector function, e.g. a molecule, which prevents or interfereswith binding and/or activation of a native sequence ErbB receptor by aligand, and/or downstream pathways used by the native sequence ErbBreceptor. Such molecules can be screened, for example, based upon theirability to competitively inhibit ErbB receptor activation by ligand inthe tyrosine phosphorylation assay. Similarly, an antagonist of a nativesequence ErbB4 (HER4) receptor is a molecule which prevents orinterferes with an ErbB4 effector function, e.g. a molecule whichprevents or interferes with binding and/or activation of a nativesequence ErbB4 receptor by a ligand, and/or downstream pathways used bythe ErbB4 receptor. Such molecules can be screened, for example, basedupon their ability to competitively inhibit ErbB4 receptor activation byligand in the tyrosine phosphorylation assay. Examples of ErbB4antagonists include, without limitation, soluble ErbB4 receptors (suchas extracellular domains (ECD) of native sequence and variant ErbB4receptors), neutralizing antibodies against native sequence ErbB4receptors, neutralizing antibodies to ligands of native sequence ErbB4receptors (e.g. anti-HB-EGF antibodies), ErbB4-Ig immunoadhesins(including chimeric heteroadhesins) and small molecules.

By “ErbB4 ligand” is meant a polypeptide which binds to and/or activatesan ErbB4 receptor. ErbB4 ligands include betacellulin, epiregulin,HB-EGF, NRG-2, NRG-3 and heregulins.

In the methods of the present invention, the term “control” andgrammatical variants thereof, are used to refer to the prevention,partial or complete inhibition, reduction, delay or slowing down of anunwanted event, e.g. physiological condition, such as the excessiveproliferation and/or migration of smooth muscle cells and/or other celltypes, e.g. endothelial cells.

The term “excessive proliferation and/or migration” means proliferationand/or migration beyond normal levels that results or is likely toresult, if untreated, in the development of an unwanted physiologicalcondition or disease, such as, for example, stenosis, including vascularstenosis, restenosis, and pyloric stenosis; urinary bladder wallthickening, and obstructive airway disease.

“Treatment” refers to both therapeutic treatment and prophylactic orpreventative measures. Those in need of treatment include those alreadywith the disorder as well as those prone to have the disorder or thosein which the disorder is to be prevented. For purposes of thisinvention, beneficial or desired clinical results include, but are notlimited to, alleviation of symptoms, diminishment of extent of disease,stabilized (i.e., not worsening) state of disease, delay or slowing ofdisease progression, amelioration or palliation of the disease state,and remission (whether partial or total), whether detectable orundetectable. “Treatment” can also mean prolonging survival as comparedto expected survival if not receiving treatment. Those in need oftreatment include those already with the condition or disorder as wellas those prone to have the condition or disorder or those in which thecondition or disorder is to be prevented.

The term “isolated” molecule is defined broadly as a molecule that isidentified and separated from at least one contaminant molecule withwhich it is ordinarily associated in the natural source of the molecule.Preferably, the isolated molecule is free of association with allcomponents with which it is naturally associated.

The term “immunoadhesin” as used herein refers to antibody-likemolecules that combine the binding domain of a protein such as anextracellular domain (the adhesin portion) of a cell-surface receptorwith the effector functions of an immunoglobulin constant domain. Theterm “immunoadhesin” specifically includes native or variant ErbB4receptor sequences. The nucleic acid sequence of an ErbB4-IgGimmunoadhesin is presented in FIG. 3 (SEQ ID NO: 3). Immunoadhesins canpossess many of the valuable chemical and biological properties of humanantibodies. Since immunoadhesins can be constructed from a human proteinsequence with a desired specificity linked to an appropriate humanimmunoglobulin hinge and constant domain (Fc) sequence, the bindingspecificity of interest can be achieved using entirely human components.Such immunoadhesins are minimally immunogenic to the patient, and aresafe for chronic or repeated use. The term “isolated immunoadhesin”refers to an immunoadhesin that has been purified from a source or hasbeen prepared by recombinant or synthetic methods and is sufficientlyfree of other peptides or proteins.

Immunoadhesins reported in the literature include fusions of the T cellreceptor (Gascoigne et al., Proc. Natl. Acad. Sci. USA 84:2936-2940(1987)); CD4 (Capon et al., Nature 337:525-531 (1989); Traunecker etal., Nature 339:68-70 (1989); Zettmeissl et al., DNA Cell Biol. USA9:347-353 (1990); and Byrn et al., Nature 344:667-670 (1990));L-selectin or homing receptor (Watson et al., J. Cell. Biol.110:2221-2229 (1990); and Watson et al., Nature 349:164-167 (1991));CD44 (Aruffo et al., Cell 61:1303-1313 (1990)); CD28 and B7 (Linsley etal., J. Exp. Med. 173:721-730 (1991)); CTLA-4 (Lisley et al., J. Exp.Med. 174:561-569 (1991)); CD22 (Stamenkovic et al., Cell 66:1133-1144(1991)); TNF receptor (Ashkenazi et al., Proc. Natl. Acad. Sci. USA88:10535-10539 (1991); Lesslauer et al., Eur. J. Immunol. 27:2883-2886(1991); and Peppel et al., J. Exp. Med. 174:1483-1489 (1991)); NPreceptors (Bennett et al., J. Biol. Chem. 266:23060-23067 (1991));inteferon γ receptor (Kurschner et al., J. Biol. Chem. 267:9354-9360(1992)); 4-1BB (Chalupny et al., PNAS USA 89:10360-10364 (1992)) and IgEreceptor α (Ridgway and Gorman, J. Cell. Biol. 115, Abstract No. 1448(1991)).

Examples of homomultimeric immunoadhesins which have been described fortherapeutic use include the CD4-IgG immunoadhesin for blocking thebinding of HIV to cell-surface CD4. Data obtained from Phase I clinicaltrials, in which CD4-IgG was administered to pregnant women just beforedelivery, suggests that this immunoadhesin may be useful in theprevention of maternal-fetal transfer of HIV (Ashkenazi et al., Intern.Rev. Immunol. 10:219-227 (1993). An immunoadhesin which binds tumornecrosis factor (TNF) has also been developed. TNF is a proinflammatorycytokine which has been shown to be a major mediator of septic shock.Based on a mouse model of septic shock, a TNF receptor immunoadhesin hasshown promise as a candidate for clinical use in treating septic shock(Ashkenazi, A. et al. (1991) PNAS USA 88:10535-10539). ENBREL®(etanercept), an immunoadhesin comprising a TNF receptor sequence fusedto an IgG Fc region, was approved by the U.S. Food and DrugAdministration (FDA), on Nov. 2, 1998, for the treatment of rheumatoidarthritis. The new expanded use of ENBREL® in the treatment ofrheumatoid arthritis has recently been approved by FDA on Jun. 6, 2000.For recent information on TNF blockers, including ENBREL®, see Lovell etal., N. Engl. J. Med. 342: 763-169 (2000), and accompanying editorial onp 810-811; and Weinblatt et al., N. Engl. J. Med. 340: 253-259 (1999);reviewed in Maini and Taylor, Annu. Rev. Med. 51: 207-229 (2000).Immunoadhesins also have non-therapeutic uses. For example, theL-selectin receptor immunoadhesin was used as a reagent forhistochemical staining of peripheral lymph node high endothelial venules(HEV). This reagent was also used to isolate and characterize theL-selectin ligand (Ashkenazi et al., supra).

If the two arms of the immunoadhesin structure have differentspecificities, the immunoadhesin is called a “bispecific immunoadhesin”by analogy to bispecific antibodies. Dietsch et al., J. Immunol. Methods162:123 (1993) describe such a bispecific immunoadhesin combining theextracellular domains of the adhesion molecules, E-selectin andP-selectin, each of which selectins is expressed in a different celltype in nature. Binding studies indicated that the bispecificimmunoglobulin fusion protein so formed had an enhanced ability to bindto a myeloid cell line compared to the monospecific immunoadhesins fromwhich it was derived.

The term “heteroadhesin” is used interchangeably with the expression“chimeric heteromultimer adhesin” and refers to a complex of chimericmolecules (amino acid sequences) in which each chimeric moleculecombines a biologically active portion, such as the extracellular domainof each of the heteromultimeric receptor monomers, with amultimerization domain. The “multimerization domain” promotes stableinteraction of the chimeric molecules within the heteromultimer complex.The multimerization domains may interact via an immunoglobulin sequence,leucine zipper, a hydrophobic region, a hydrophilic region, or a freethiol which forms an intermolecular disulfide bond between the chimericmolecules of the chimeric heteromultimer. The multimerization domain maycomprise an immunoglobulin constant region. In addition amultimerization region may be engineered such that steric interactionsnot only promote stable interaction, but further promote the formationof heterodimers over homodimers from a mixture of monomers.“Protuberances” are constructed by replacing small amino acid sidechains from the interface of the first polypeptide with larger sidechains (e.g. tyrosine or tryptophan). Compensatory “cavities” ofidentical or similar size to the protuberances are optionally created onthe interface of the second polypeptide by replacing large amino acidside chains with smaller ones (e.g. alanine or threonine). Theimmunoglobulin sequence preferably, but not necessarily, is animmunoglobulin constant domain. The immunoglobulin moiety in thechimeras of the present invention may be obtained from IgG₁, IgG₂, IgG₃or IgG₄ subtypes, IgA, IgE, IgD or IgM, but preferably IgG₁ or IgG₃.

The term “epitope tagged” when used herein refers to a chimericpolypeptide comprising the entire chimeric heteroadhesin, or a fragmentthereof, fused to a “tag polypeptide”. The tag polypeptide has enoughresidues to provide an epitope against which an antibody can be made,yet is short enough such that it does not interfere with activity of thechimeric heteroadhesin. The tag polypeptide preferably is fairly uniqueso that the antibody thereagainst does not substantially cross-reactwith other epitopes. Suitable tag polypeptides generally have at least 6amino acid residues and usually between about 8-50 amino acid residues(preferably between about 9-30 residues). An embodiment of the inventionencompasses a chimeric heteroadhesin linked to an epitope tag, which tagis used to detect the adhesin in a sample or recover the adhesin from asample.

“Isolated/highly purified/substantially homogenous immunoadhesin”,“isolated/highly purified/substantially homogenous heteroadhesin”, and“isolated/highly purified/substantially homogenous chimericheteromultimer adhesin”, are used interchangeably and mean the adhesinthat has been purified from a source or has been prepared by recombinantor synthetic methods and is sufficiently free of other peptides orproteins to homogeneity by chromatographic techniques or otherpurification techniques, such as SDS-PAGE under non-reducing or reducingconditions using Coomassie blue or, preferably, silver stain.Homogeneity here means less than about 5% contamination with othersource proteins. The ErbB2/4-IgG chimeric heteroadhesins of theinvention bind with sufficiently greater affinity relative to thehomodimers that the use of a mixture of homodimers and heterodimers isalso considered a useful embodiment of the invention. The terms“chimeric heteromultimer adhesin”, “chimeric heteroadhesin” and “CHA”are used interchangeably herein.

The term “antibody” is used in the broadest sense and specificallycovers antibodies that recognize native ErbB4 receptors. An antibodythat shows “high affinity” binding has a Kd of less than about 100 nM,preferably less than about 50, more preferably less than about 25, mostpreferably less than about 10.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally-occurring mutations that may be present inminor amounts. Monoclonal antibodies are highly specific, being directedagainst a single antigenic site. Furthermore, in contrast toconventional (polyclonal) antibody preparations which typically includedifferent antibodies directed against different determinants (epitopes),each monoclonal antibody is directed against a single determinant on theantigen.

The monoclonal antibodies herein include hybrid and recombinantantibodies produced by splicing a variable (including hypervariable)domain of an anti-chimeric heteroadhesin antibody with a constant domain(e.g. “humanized” antibodies), or a light chain with a heavy chain, or achain from one species with a chain from another species, or fusionswith heterologous proteins, regardless of species of origin orimmunoglobulin class or subclass designation, as well as antibodyfragments (e.g., Fab, F(ab)₂, and Fv), so long as they exhibit thedesired biological activity. (See, e.g., U.S. Pat. No. 4,816,567 andMage & Lamoyi, in Monoclonal Antibody Production Techniques andApplications, pp. 79-97 (Marcel Dekker, Inc.), New York (1987)).

Thus, the modifier “monoclonal” indicates the character of the antibodyas being obtained from a substantially homogeneous population ofantibodies, and is not to be construed as requiring production of theantibody by any particular method. For example, the monoclonalantibodies to be used in accordance with the present invention may bemade by the hybridoma method first described by Kohler & Milstein,Nature 256:495 (1975), or may be made by recombinant DNA methods (U.S.Pat. No. 4,816,567). The “monoclonal antibodies” may also be isolatedfrom phage libraries generated using the techniques described inMcCafferty et al., Nature 348:552-554 (1990), for example.

“Humanized” forms of non-human (e.g. murine) antibodies are specificchimeric immunoglobulins, immunoglobulin chains or fragments thereof(such as Fv, Fab, Fab′, F(ab)₂ or other antigen-binding subsequences ofantibodies) which contain minimal sequence derived from non-humanimmunoglobulin. For the most part, humanized antibodies are humanimmunoglobulins (recipient antibody) in which residues from thecomplementarity determining regions (CDRs) of the recipient antibody arereplaced by residues from the CDRs of a non-human species (donorantibody) such as mouse, rat or rabbit having the desired specificity,affinity and capacity. In some instances, Fv framework region (FR)residues of the human immunoglobulin are replaced by correspondingnon-human FR residues. Furthermore, the humanized antibody may compriseresidues which are found neither in the recipient antibody nor in theimported CDR or FR sequences. These modifications are made to furtherrefine and optimize antibody performance. In general, the humanizedantibody will comprise substantially all of at least one, and typicallytwo, variable domains, in which all or substantially all of the CDRregions correspond to those of a non-human immunoglobulin and all orsubstantially all of the FR residues are those of a human immunoglobulinconsensus sequence. The humanized antibody optimally also will compriseat least a portion of an immunoglobulin constant region (Fc), typicallythat of a human immunoglobulin.

“Muscle cells” include skeletal, cardiac or smooth muscle tissue cells.This term encompasses those cells which differentiate to form morespecialized muscle cells (e.g. myoblasts). Vascular smooth muscle cellsrefer to smooth muscle cells present in a middle elastic layer, media,of blood vessels.

The term “stenosis” refers to narrowing or stricture of a hollow passage(e,g, a duct or canal) in the body. The term “vascular stenosis” refersto occlusion or narrowing of blood vessels. Vascular stenosis oftenresults from fatty deposit (as in the case of atherosclerosis) orexcessive migration and proliferation of vascular smooth muscle cellsand endothelial cells. Arteries are particularly susceptible tostenosis. The term “stenosis” as used herein specifically includesinitial stenosis and restenosis.

The term “restenosis” refers to recurrence of stenosis after treatmentof initial stenosis with apparent success. For example, “restenosis” inthe context of vascular stenosis, refers to the reoccurrence of vascularstenosis after it has been treated with apparent success, e.g. byremoval of fatty deposit by balloon angioplasty. One of the contributingfactors in restenosis is intimal hyperplasia. The term “intimalhyperplasia”, used interchangeably with “neointimal hyperplasia” and“neointima formation”, refers to thickening of the inner most layer ofblood vessels, intima, as a consequence of excessive proliferation andmigration of vascular smooth muscle cells and endothelial cells. Thevarious changes taking place during restenosis are often collectivelyreferred to as “vascular wall remodeling.”

The terms “balloon angioplasty” and “percutaneous transluminal coronaryangioplasty” (PTCA) are often used interchangeably, and refer to anon-surgical catheter-based treatment for removal of plaque from thecoronary artery. Stenosis or restenosis often lead to hypertension as aresult of increased resistance to blood flow.

The term “pyloric stenosis” refers to narrowing of pylorus, the passageat the lower end of the stomach that opens into the duodenum.

The term “hypertension” refers to abnormally high blood pressure, i.e.beyond the upper value of the normal range.

By “neutralizing antibody” is meant an antibody molecule as hereindefined which is able to block or significantly reduce an effectorfunction of ErbB receptors. Accordingly, a “neutralizing” anti-ErbB4antibody is capable of blocking or significantly reducing an effectorfunction, such as ligand binding and/or elicitation of a cellularresponse, of ErbB4. For the purpose of the present invention, theability of an anti-ErbB4 antibody to neutralize the binding of an ErbB4ligand (heregulin, HRG) to ErbB4 can be monitored, for example, bymeasuring the binding of detectably labeled HRG to purified ErbB4 or toa cell line expressing or modified to express ErbB4 in the presence andabsence of a candidate anti-ErbB4 antibody. Such assays are described inExample 4 below. For the purpose of the present invention, the abilityof the anti-ErbB4 antibodies to neutralize the elicitation of a cellularresponse by ErbB4 is preferably tested by monitoring the inhibition oftyrosine phosphorylation of ErbB4 by heregulin (HRG), or in a cellproliferation assay. Representative assays are disclosed in Example 4below. “Significant” reduction means at least about 60%, or at leastabout 70%, preferably at least about 75%, more preferably at least about80%, even more preferably at least about 85%, still more preferably atleast about 90%, still more preferably at least about 95%, mostpreferably at least about 99% reduction of an effector function of thetarget antigen (e.g. ErbB4), such as ligand (e.g. HRG) binding and/orelicitation of a cellular response. Preferably, the “neutralizing”antibodies as defined herein will be capable of neutralizing at leastabout 60%, or at least about 70%, preferably at least about 75%, morepreferably at least about 80%; even more preferably at least about 85%,still more preferably at least about 90%, still more preferably at leastabout 95%, most preferably at least about 99% of the tyrosinephosphorylation of ErbB4 by HRG, as determined by the assay described inExample 4.

An “isolated” antibody is one that has been identified and separatedand/or recovered from a component of its natural environment.Contaminant components of its natural environment are materials thatwould interfere with diagnostic or therapeutic uses for the antibody,and may include enzymes, hormones, and other proteinaceous ornon-proteinaceous solutes. In preferred embodiments, the antibody willbe purified (1) to greater than 95% by weight of antibody as determinedby the Lowry method, and most preferably more than 99% by weight, (2) toa degree sufficient to obtain at least 15 residues of N-terminal orinternal amino acid sequence by use of a spinning cup sequenator, or (3)to homogeneity by SDS-PAGE under reducing or non-reducing conditionsusing Coomassie blue or, preferably, silver stain. Isolated antibodyincludes the antibody in situ within recombinant cells since at leastone component of the antibody's natural environment will not be present.Ordinarily, however, isolated antibody will be prepared by at least onepurification step.

The term “epitope” is used to refer to binding sites for (monoclonal orpolyclonal) antibodies on protein antigens.

Antibodies which bind to a particular epitope can be identified by“epitope mapping.” There are many methods known in the art for mappingand characterizing the location of epitopes on proteins, includingsolving the crystal structure of an antibody-antigen complex,competition assays, gene fragment expression assays, and syntheticpeptide-based assays, as described, for example, in Chapter 11 of Harlowand Lane, Using Antibodies, a Laboratory Manual, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1999. Competition assays arediscussed below. According to the gene fragment expression assays, theopen reading frame encoding the protein is fragmented either randomly orby specific genetic constructions and the reactivity of the expressedfragments of the protein with the antibody to be tested is determined.The gene fragments may, for example, be produced by PCR and thentranscribed and translated into protein in vitro, in the presence ofradioactive amino acids. The binding of the antibody to theradioactively labeled protein fragments is then determined byimmunoprecipitation and gel electrophoresis. Certain epitopes can alsobe identified by using large libraries of random peptide sequencesdisplayed on the surface of phage particles (phage libraries).Alternatively, a defined library of overlapping peptide fragments can betested for binding to the test antibody in simple binding assays. Thelatter approach is suitable to define linear epitopes of about 5 to 15amino acids.

An antibody binds “essentially the same epitope” as a referenceantibody, when the two antibodies recognize identical or stericallyoverlapping epitopes. The most widely used and rapid methods fordetermining whether two epitopes bind to identical or stericallyoverlapping epitopes are competition assays, which can be configured inall number of different formats, using either labeled antigen or labeledantibody. Usually, the antigen is immobilized on a 96-well plate, andthe ability of unlabeled antibodies to block the binding of labeledantibodies is measured using radioactive or enzyme labels.

The phrase “inhibiting an ErbB4 (HER4) receptor” refers to the abilityof an ErbB4 antagonist to inhibit or prevent activation of an ErbB4receptor, for example, by blocking the binding of a ligand to the ErbB4receptor. The “activation” of an ErbB4 receptor refers to receptorphosphorylation, which can be quantified using the tyrosinephosphorylation assays, and downstream events that constitute inductionof signal transduction by the bound ligand. “Inhibition” is any of theseassays is at least about 60%, or at least about 70%, preferably at leastabout 75%, more preferably at least about 80%; even more preferably atleast about 85%, still more preferably at least about 90%, still morepreferably at least about 95%, most preferably at least about 99%.

The expression “decreasing survival of a cell” refers to the act ofdecreasing the period of existence of a cell, relative to an untreatedcell which has not been exposed to a ERbB4 antagonist either in vitro orin vivo. The expression “decreased cell proliferation” refers to adecrease in the number of cells in a population exposed to an ErbB4antagonist either in vitro or in vivo, relative to an untreated cell.

“Biological activity” where used in conjunction with an ErbB4 antagonistrefers to the ability of an ErbB4 antagonist to control the excessiveproliferation or migration of smooth muscle cells, as determined in arelevant in vitro or in vivo assay, including the PDGF-stimulated smoothmuscle cell proliferation and human aortic smooth muscle cell migrationassays described in the Examples hereinbelow, animal models and humanclinical trials, irrespective of the underlying mechanism. Thus, thebiological activity of an ErbB4 antagonist includes, without limitation,functioning as an inhibitor of the binding of a ligand or activation ofa native ErbB4 receptor, and/or inhibition of growth and/or migration ofsmooth muscle cells expressing an ErbB4 receptor on their surface.

The term “disease state” refers to a physiological state of a cell or ofa whole mammal in which an interruption, cessation, or disorder ofcellular or body functions systems, or organs has occurred.

The term “effective amount” refers to an amount of a drug effective totreat (including prevention) a disease, disorder or unwantedphysiological conditions in a mammal. In the present invention, an“effective amount” of an ErbB4 antagonist may reduce, slow down or delaythe proliferation of smooth muscle cells; reduce, slow down or delay themigration of smooth muscle cells; prevent or inhibit (i.e., slow to someextent and preferably stop) the development of stenosis or restenosis;and/or relieve to some extent one or more of the symptoms associatedwith stenosis or restenosis, in particular, prevent or inhibit (i.e.,slow to some extent and preferably stop) the development of elevatedblood pressure associated with stenosis or restenosis.

Nucleic acid is “operably linked” when it is placed into a functionalrelationship with another nucleic acid sequence. For example, DNA for apresequence or secretory leader is operably linked to DNA for apolypeptide if it is expressed as a preprotein that participates in thesecretion of the polypeptide; a promoter or enhancer is operably linkedto a coding sequence if it affects the transcription of the sequence; ora ribosome binding site is operably linked to a coding sequence if it ispositioned so as to facilitate translation. Generally, “operably linked”means that the DNA sequences being linked are contiguous, and, in thecase of a secretory leader, contiguous and in reading phase. However,enhancers do not have to be contiguous. Linking is accomplished byligation at convenient restriction sites. If such sites do not exist,the synthetic oligonucleotide adapters or linkers are used in accordancewith conventional practice.

“Pharmaceutically acceptable” carriers, excipients, or stabilizers areones which are nontoxic to the cell or mammal being exposed thereto atthe dosages and concentrations employed. Often the physiologicallyacceptable carrier is an aqueous pH buffered solution. Examples ofphysiologically acceptable carriers include buffers such as phosphate,citrate, and other organic acids; antioxidants including ascorbic acid;low molecular weight (less than about 10 residues) polypeptides;proteins, such as serum albumin, gelatin, or immunoglobulins;hydrophilic polymers such as polyvinylpyrrolidone; amino acids such asglycine, glutamine, asparagine, arginine or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugar alcohols such as mannitolor sorbitol; salt-forming counterions such as sodium; and/or nonionicsurfactants such as Tween™, polyethylene glycol (PEG), and Pluronics™.

B. Methods for Carrying Out the Invention

The invention concerns the treatment of stenosis by antagonists ofnative ErbB4 receptors. Although the invention is not so limited, in apreferred embodiment, the antagonist is an immunoadhesin or a chimericheteromultimer adhesin. Immunoadhesins (referred to as hybridimmunoglobulins), including their structure and preparation, aredescribed, e.g. in WO 91/08298; and in U.S. Pat. Nos. 5,428,130 and5,116,964, the disclosures of which are hereby expressly incorporated byreference.

1. Production of an Immunoadhesin or Chimeric Heteromultimer Adhesin.

The description below relates primarily to production of immunoadhesinby culturing cells transformed or transfected with a vector containingimmunoadhesin nucleic acid. It is, of course, contemplated thatalternative methods, which are well known in the art, may be employed toprepare immunoadhesin. For instance, the immunoadhesin sequence, orportions thereof, may be produced by direct peptide synthesis usingsolid-phase techniques [see, e.g., Stewart et al., Solid-Phase PeptideSynthesis, W.H. Freeman Co., San Francisco, Calif. (1969); Merrifield,J. Am. Chem. Soc., 85:2149-2154 (1963)]. In vitro protein synthesis maybe performed using manual techniques or by automation. Automatedsynthesis may be accomplished, for instance, using an Applied BiosystemsPeptide Synthesizer (Foster City, Calif.) using manufacturer'sinstructions. Various portions of the immunoadhesin may be chemicallysynthesized separately and combined using chemical or enzymatic methodsto produce the full-length immunoadhesin.

Nucleic acid encoding a native sequence ErbB4 receptor can, for example,be isolated from cells known to express the ErbB4 receptor, such asthose described in EP 599,274, supra, and in the collective Plowman etal. references, supra or is synthesized.

DNA encoding immunoglobulin light or heavy chain constant regions isknown or readily available from cDNA libraries or is synthesized. Seefor example, Adams et al., Biochemistry 19:2711-2719 (1980); Gough etal., Biochemistry 19:2702-2710 (1980); Dolby et al; P.N.A.S. USA,77:6027-6031 (1980); Rice et al P.N.A.S USA 79:7862-7865 (1982); Falkneret al; Nature 298:286-288 (1982); and Morrison et al; Ann. Rev. Immunol.2:239-256 (1984).

An immunoadhesin or a chimeric heteroadhesin of the invention ispreferably produced by expression in a host cell and isolated therefrom.A host cell is generally transformed with the nucleic acid of theinvention. Preferably the nucleic acid is incorporated into anexpression vector. Suitable host cells for cloning or expressing thevectors herein are prokaryote host cells (such as E. coli, strains ofBacillus, Pseudomonas and other bacteria), yeast and other eukaryoticmicrobes, and higher eukaryote cells (such as Chinese hamster ovary(CHO) cells and other mammalian cells). The cells may also be present inlive animals (for example, in cows, goats or sheep). Insect cells mayalso be used. Cloning and expression methodologies are well known in theart.

To obtain expression of an immunoadhesin such as a chimeric ErbB4-IgGmolecule, one or more expression vector(s) is/are introduced into hostcells by transformation or transfection and the resulting recombinanthost cells are cultured in conventional nutrient media, modified asappropriate for inducing promoters, selecting recombinant cells, oramplifying the ErbB4-IgG DNA. In general, principles, protocols, andpractical techniques for maximizing the productivity of in vitromammalian cell cultures can be found in Mammalian Cell Biotechnology: aPractical Approach, M. Butler, ed. (IRL Press, 1991).

(i) Construction of Nucleic Acid Encoding Immunoadhesin

When preparing the immunoadhesins of the present invention, preferablynucleic acid encoding an extracellular domain of a natural receptor isfused C-terminally to nucleic acid encoding the N-terminus of animmunoglobulin constant domain sequence, however N-terminal fusions arealso possible. Typically, in such fusions the encoded chimericpolypeptide will retain at least functionally active hinge, CH2 and CH3domains of the constant region of an immunoglobulin heavy chain. Fusionsare also made to the C-terminus of the Fc portion of a constant domain,or immediately N-terminal to the CH1 of the heavy chain or thecorresponding region of the light chain. The resultant DNA fusionconstruct is expressed in appropriate host cells.

Nucleic acid molecules encoding amino acid sequence variants of nativesequence extracellular domains (such as from ErbB4) and/or the antibodysequences used to prepare the desired immunoadhesin, are prepared by avariety of methods known in the art. These methods include, but are notlimited to, isolation from a natural source (in the case of naturallyoccurring amino acid sequence variants, such as those mentioned above inconnection with ErbB4) or preparation by oligonucleotide-mediated (orsite-directed) mutagenesis, PCR mutagenesis, and cassette mutagenesis ofan earlier prepared variant or a non-variant version of native sequenceErbB4.

Amino acid sequence variants of native sequence extracellular domainincluded in the chimeric heteroadhesin are prepared by introducingappropriate nucleotide changes into the native extracellular domain DNAsequence, or by in vitro synthesis of the desired chimeric heteroadhesinmonomer polypeptide. Such variants include, for example, deletions from,or insertions or substitutions of, residues in the amino acid sequenceof the immunoadhesin or chimeric heteroadhesin.

Variations in the native sequence as described above can be made usingany of the techniques and guidelines for conservative andnon-conservative mutations set forth in Table 1.

In a preferred embodiment, the nucleic acid encodes a chimeric moleculein which the ErbB4 receptor extracellular domain sequence is fused tothe N-terminus of the C-terminal portion of an antibody (in particularthe Fc domain), containing the effector functions of an immunoglobulin,e.g. IgG1. It is possible to fuse the entire heavy chain constant regionto the ErbB4 receptor extracellular domain sequence. However, morepreferably, a sequence beginning in the hinge region just upstream ofthe papain cleavage site (which defines IgG Fc chemically; residue 216,taking the first residue of heavy chain constant region to be 114 [Kobetet al., supra], or analogous sites of other immunoglobulins) is used inthe fusion. In a particularly preferred embodiment, the ErbB4 receptorextracellular domain sequence is fused to the hinge region and CH2 andCH3 or CHI, hinge, CH2 and CH3 domains of an IgG1, IgG2, or IgG3 heavychain. The precise site at which the fusion is made is not critical, andthe optimal site can be determined by routine experimentation.

For human immunoadhesins, the use of human IgG1 and IgG3 immunoglobulinsequences is preferred. A major advantage of using IgG1 is that IgG1immunoadhesins can be purified efficiently on immobilized protein A. Incontrast, purification of IgG3 requires protein G, a significantly lessversatile medium. However, other structural and functional properties ofimmunoglobulins should be considered when choosing the Ig fusion partnerfor a particular immunoadhesin construction. For example, the IgG3 hingeis longer and more flexible, so it can accommodate larger “adhesin”domains that may not fold or function properly when fused to IgG1.Another consideration may be valency; IgG immunoadhesins are bivalenthomodimers, whereas Ig subtypes like IgA and IgM may give rise todimeric or pentameric structures, respectively, of the basic Ighomodimer unit.

For ErbB4-Ig immunoadhesins designed for in vivo application, thepharmacokinetic properties and the effector functions specified by theFc region are important as well. Although IgG1, IgG2 and IgG4 all havein vivo half-lives of 21 days, their relative potencies at activatingthe complement system are different. IgG4 does not activate complement,and IgG2 is significantly weaker at complement activation than IgG1.Moreover, unlike IgG1, IgG2 does not bind to Fc receptors on mononuclearcells or neutrophils. While IgG3 is optimal for complement activation,its in vivo half-life in approximately one third of the other IgGisotypes.

Another important consideration for immunoadhesins designed to be usedas human therapeutics is the number of allotypic variants of theparticular isotype. In general, IgG isotypes with fewerserologically-defined allotypes are preferred. For example, IgG1 hasonly four serologically-defined allotypic sites, two of which (G1 m and2) are located in the Fc region; and one of these sites G1m1, isnon-immunogenic. In contrast, there are 12 serologically-definedallotypes in IgG3, all of which are in the Fc region; only three ofthese sites (G3m5, 11 and 21) have one allotype which is nonimmunogenic.Thus, the potential immunogenicity of an IgG3 immunoadhesin is greaterthan that of an IgG1 immunoadhesin.

The cDNAs encoding the ErbB4 receptor sequence (e.g. an extracellulardomain sequence) and the Ig parts of the immunoadhesin are inserted intandem into a plasmid vector that directs efficient expression in thechosen host cells. For expression in mammalian cells pRK5-based vectors[Schall et al., Cell 61, 361-370 (1990)] and CDM8-based vectors [Seed,Nature 329, 840 (1989)] may, for example, be used. The exact junctioncan be created by removing the extra sequences between the designedjunction codons using oligonucleotide-directed deletional mutagenesis[Zoller and Smith, Nucleic Acids Res. 10, 6487 (1982); Capon et al.,Nature 337, 525-531 (1989)]. Synthetic oligonucleotides can be used, inwhich each half is complementary to the sequence on either side of thedesired junction; ideally, these are 36 to 48-mers. Alternatively, PCRtechniques can be used to join the two parts of the molecule in-framewith an appropriate vector.

Although the presence of an immunoglobulin light chain is not requiredin the immunoadhesins of the present invention, an immunoglobulin lightchain might be present either covalently associated to an trkreceptor-immunoglobulin heavy chain fusion polypeptide, or directlyfused to the trk receptor extracellular domain. In the former case, DNAencoding an immunoglobulin light chain is typically coexpressed with theDNA encoding the ErbB4 receptor-immunoglobulin heavy chain fusionprotein. Upon secretion, the hybrid heavy chain and the light chain willbe covalently associated to provide an immunoglobulin-like structurecomprising two disulfide-linked immunoglobulin heavy chain-light chainpairs. Method suitable for the preparation of such structures are, forexample, disclosed in U.S. Pat. No. 4,816,567 issued Mar. 28, 1989.

Another preferred type of chimeric ErbB4 antagonist herein is a fusionprotein comprising an extracellular domain, such as from a ErbB4monomer, linked to a heterologous polypeptide, such as a multimerizationdomain. Such a sequence can be constructed using recombinant DNAtechniques. Alternatively, the heterologous polypeptide can becovalently bound to the extracellular domain polypeptide by techniqueswell known in the art such as the use of the heterobifunctionalcrosslinking reagents. Exemplary coupling agents includeN-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane(IT), bifunctional derivatives of imidoesters (such as dimethyladipimidate HCL), active esters (such as disuccinimidyl suberate),aldehydes (such as glutareldehyde), bis-azido compounds (such as bis(p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such asbis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene).

In one embodiment, a chimeric heteroadhesin polypeptide comprises afusion of a monomer of the chimeric heteroadhesin with a tag polypeptidewhich provides an epitope to which an anti-tag antibody can selectivelybind. Such epitope tagged forms of the chimeric heteroadhesin areuseful, as the presence thereof can be detected using a labeled antibodyagainst the tag polypeptide. Also, provision of the epitope tag enablesthe chimeric heteroadhesin to be readily purified by affinitypurification using the anti-tag antibody. Tag polypeptides and theirrespective antibodies are well known in the art. Examples include theflu HA tag polypeptide and its antibody 12CA5, (Field et al., Mol. Cell.Biol. 8:2159-2165 (1988)); the c-myc tag and the 8F9, 3C7, 6E10, G4, B7and 9E10 antibodies thereto (Evan et al., Molecular and Cellular Biology5(12):3610-3616 (1985)); and the Herpes Simplex virus glycoprotein D(gD) tag and its antibody (Paborsky et al., Protein Engineering3(6):547-553 (1990)).

Another type of covalent modification of a chimeric heteromultimercomprises linking a monomer polypeptide of the heteromultimer to one ofa variety of non-proteinaceous polymers, e.g., polyethylene glycol,polypropylene glycol, polyoxyalkylenes, or copolymers of polyethyleneglycol and polypropylene glycol. A chimeric heteromultimer also may beentrapped in microcapsules prepared, for example, by coacervationtechniques or by interfacial polymerization (for example,hydroxymethylcellulose or gelatin-microcapsules andpoly-(methylmethacylate) microcapsules, respectively), in colloidal drugdelivery systems (for example, liposomes, albumin microspheres,microemulsions, nano-particles and nanocapsules), or in macroemulsions.Such techniques are disclosed in Remington's Pharmaceutical Sciences,16th edition, Oslo, A., Ed., (1980).

(ii) Selection and Transformation of Host Cells

Host cells are transfected or transformed with expression or cloningvectors described herein for immunoadhesin production and cultured inconventional nutrient media modified as appropriate for inducingpromoters, selecting transformants, or amplifying the genes encoding thedesired sequences. The culture conditions, such as media, temperature,pH and the like, can be selected by the skilled artisan without undueexperimentation. In general, principles, protocols, and practicaltechniques for maximizing the productivity of cell cultures can be foundin Mammalian Cell Biotechnology: a Practical Approach, M. Butler, ed.(IRL Press, 1991) and Sambrook et al., supra.

The terms “transformation” and “transfection” are used interchangeablyherein and refer to the process of introducing DNA into a cell.Following transformation or transfection, the nucleic acid of theinvention may integrate into the host cell genome, or may exist as anextrachromosomal element. Methods of eukaryotic cell transfection andprokaryotic cell transformation are known to the ordinarily skilledartisan, for example, CaCl₂, CaPO₄, liposome-mediated andelectroporation. Depending on the host cell used, transformation isperformed using standard techniques appropriate to such cells. Thecalcium treatment employing calcium chloride, as described in Sambrooket al., supra, or electroporation is generally used for prokaryotes.Infection with Agrobacterium tumefaciens is used for transformation ofcertain plant cells, as described by Shaw et al., Gene, 23:315 (1983)and WO 89/05859 published 29 Jun. 1989. For mammalian cells without suchcell walls, the calcium phosphate precipitation method of Graham and vander Eb, Virology, 52:456-457 (1978) can be employed. General aspects ofmammalian cell host system transfections have been described in U.S.Pat. No. 4,399,216. Transformations into yeast are typically carried outaccording to the method of Van Solingen et al., J. Bact., 130:946 (1977)and Hsiao et al., Proc. Natl. Acad. Sci. (USA), 76:3829 (1979). However,other methods for introducing DNA into cells, such as by nuclearmicroinjection, electroporation, bacterial protoplast fusion with intactcells, or polycations, e.g., polybrene, polyornithine, may also be used.For various techniques for transforming mammalian cells, see Keown etal., Methods in Enzymology, 185:527-537 (1990) and Mansour et al.,Nature, 336:348-352 (1988).

Suitable host cells for cloning or expressing the DNA in the vectorsherein include prokaryote, yeast, or higher eukaryote cells. Suitableprokaryotes include but are not limited to eubacteria, such asGram-negative or Gram-positive organisms, for example,Enterobacteriaceae such as E. coli. Various E. coli strains are publiclyavailable, such as E. coli K12 strain MM294 (ATCC 31,446); E. coli X1776(ATCC 31,537); E. coli strain W3110 (ATCC 27,325) and K5772 (ATCC53,635). Other suitable prokaryotic host cells includeEnterobacteriaceae such as Escherichia, e.g., E. coli, Enterobacter,Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium,Serratia, e.g., Serratia marcescans, and Shigella, as well as Bacillisuch as B. subtilis and B. licheniformis (e.g., B. licheniformis 41Pdisclosed in DD 266,710 published 12 Apr. 1989), Pseudomonas such as P.aeruginosa, and Streptomyces. These examples are illustrative ratherthan limiting. Strain W3110 is one particularly preferred host or parenthost because it is a common host strain for recombinant DNA productfermentation. Preferably, the host cell secretes minimal amounts ofproteolytic enzymes. For example, strain W3110 may be modified to effecta genetic mutation in the genes encoding proteins endogenous to thehost, with examples of such hosts including E. coli W3110 strain 1A2,which has the complete genotype tonA; E. coli W3110 strain 9E4, whichhas the complete genotype tonA ptr3; E. coli W3110 strain 27C7 (ATCC55,244), which has the complete genotype tonA ptr3 phoA E15 (argF-lac)169 degP ompT kan^(r) ; E. coli W3110 strain 37D6, which has thecomplete genotype tonA ptr3 phoA E15 (argF-lac) 169 degP ompT rbs7 ilvGkan^(r) , E. coli W3110 strain 40B4, which is strain 37D6 with anon-kanamycin resistant degP deletion mutation; and an E. coli strainhaving mutant periplasmic protease disclosed in U.S. Pat. No. 4,946,783issued 7 Aug. 1990. Alternatively, in vitro methods of cloning, e.g.,PCR or other nucleic acid polymerase reactions, are suitable.

In addition to prokaryotes, eukaryotic microbes such as filamentousfungi or yeast are suitable cloning or expression hosts forimmunoadhesin-encoding vectors. Saccharomyces cerevisiae is a commonlyused lower eukaryotic host microorganism. Others includeSchizosaccharomyces pombe (Beach and Nurse, Nature, 290: 140 [1981]; EP139,383 published 2 May 1985); Kluyveromyces hosts (U.S. Pat. No.4,943,529; Fleer et al., Bio/Technology, 9:968-975 (1991)) such as,e.g., K. lactis (MW98-8C, CBS683, CBS4574; Louvencourt et al., J.Bacteriol., 154(2): 737-1742 [1983]), K. fragilis (ATCC 12,424), K.bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24,178), K. waltii (ATCC56,500), K. drosophilarum (ATCC 36,906; Van den Berg et al.,Bio/Technology, 8:135 (1990)), K. thermotolerans, and K. marxianus;yarrowia (EP 402,226); Pichia pastoris (EP 183,070; Sreekrishna et al.,J. Basic Microbiol., 28:265-278 [1998]); Candida, Trichoderma reesia (EP244,234); Neurospora crassa (Case et al., Proc. Natl. Acad. Sci. USA,76:5259-5263 [1979]); Schwanniomyces such as Schwanniomyces occidentalis(EP 394,538 published 31 Oct. 1990); and filamentous fungi such as,e.g., Neurospora, Penicillium, Tolypocladium (WO 91/00357 published 10Jan. 1991), and Aspergillus hosts such as A. nidulans (Ballance et al.,Biochem. Biophys. Res. Commun., 112:284-289 [1983]; Tilburn et al.,Gene, 26:205-221 [1983]; Yelton et al., Proc. Natl. Acad. Sci. USA, 81:1470-1474 [1984]) and A. niger (Kelly and Hynes, EMBO J., 4:475-479[1985]). Methylotropic yeasts are suitable herein and include, but arenot limited to, yeast capable of growth on methanol selected from thegenera consisting of Hansenula, Candida, Kloeckera, Pichia,Saccharomyces, Torulopsis, and Rhodotorula. A list of specific speciesthat are exemplary of this class of yeasts may be found in C. Anthony,The Biochemistry of Methylotrophs, 269 (1982).

Suitable host cells for the expression of glycosylated immunoadhesin arederived from multicellular organisms. Examples of invertebrate cellsinclude insect cells such as Drosophila S2 and Spodoptera Sf9, as wellas plant cells. Examples of useful mammalian host cell lines includeChinese hamster ovary (CHO) and COS cells. More specific examplesinclude monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL1651); human embryonic kidney line (293 or 293 cells subcloned forgrowth in suspension culture, Graham et al., J. Gen Virol., 36:59(1977)); Chinese hamster ovary cells/-DHFR (CHO, Urlaub and Chasin,Proc. Natl. Acad. Sci. USA, 77:4216 (1980)); mouse sertoli cells (TM4,Mather, Biol. Reprod., 23:243-251 (1980)); human lung cells (W138, ATCCCCL 75); human liver cells (Hep G2, HB 8065); and mouse mammary tumor(MMT 060562, ATCC CCL51). The selection of the appropriate host cell isdeemed to be within the skill in the art.

In general, the choice of a mammalian host cell line for the expressionof ErbB4-Ig immunoadhesins depends mainly on the expression vector (seebelow). Another consideration is the amount of protein that is required.Milligram quantities often can be produced by transient transfections.For example, the adenovirus EIA-transformed 293 human embryonic kidneycell line can be transfected transiently with pRK5-based vectors by amodification of the calcium phosphate method to allow efficientimmunoadhesin expression. CDM8-based vectors can be used to transfectCOS cells by the DEAE-dextran method (Aruffo et al., Cell 61, 1303-1313(1990)]; Zettmeissl et al., DNA Cell Biol. (US) 9, 347-353 (1990)]. Iflarger amounts of protein are desired, the immunoadhesin can beexpressed after stable transfection of a host cell line. For example, apRK5-based vector can be introduced into Chinese hamster ovary (CHO)cells in the presence of an additional plasmid encoding dihydrofolatereductase (DHFR) and conferring resistance to G418. Clones resistant toG418 can be selected in culture; these clones are grown in the presenceof increasing levels of DHFR inhibitor methotrexate; clones areselected, in which the number of gene copies encoding the DHFR andimmunoadhesin sequences is co-amplified. If the immunoadhesin contains ahydrophobic leader sequence at its N-terminus, it is likely to beprocessed and secreted by the transfected cells. The expression ofimmunoadhesins with more complex structures may require uniquely suitedhost cells; for example, components such as light chain or J chain maybe provided by certain myeloma or hybridoma cell hosts [Gascoigne etal., 1987, supra; Martin et al., J. Virol. 67, 3561-3568 (1993)].

(iii) Selection and Use of a Replicable Vector

The nucleic acid encoding immunoadhesin may be inserted into areplicable vector for cloning (amplification of the DNA) or forexpression. Various vectors are publicly available. The vector may, forexample, be in the form of a plasmid, cosmid, viral particle, or phage.The appropriate nucleic acid sequence may be inserted into the vector bya variety of procedures. In general, DNA is inserted into an appropriaterestriction endonuclease site(s) using techniques known in the art.Vector components generally include, but are not limited to, one or moreof a signal sequence, an origin of replication, one or more markergenes, an enhancer element, a promoter, and a transcription terminationsequence. Construction of suitable vectors containing one or more ofthese components employs standard ligation techniques which are known tothe skilled artisan.

The immunoadhesin may be produced recombinantly not only directly, butalso as a fusion polypeptide with a heterologous polypeptide, which maybe a signal sequence or other polypeptide having a specific cleavagesite at the N-terminus of the mature protein or polypeptide. In general,the signal sequence may be a component of the vector, or it may be apart of the immunoadhesin-encoding DNA that is inserted into the vector.The signal sequence may be a prokaryotic signal sequence selected, forexample, from the group of the alkaline phosphatase, penicillinase, lpp,or heat-stable enterotoxin II leaders. For yeast secretion the signalsequence may be, e.g., the yeast invertase leader, alpha factor leader(including Saccharomyces and Kluyveromyces α-factor leaders, the latterdescribed in U.S. Pat. No. 5,010,182), or acid phosphatase leader, theC. albicans glucoamylase leader (EP 362,179 published 4 Apr. 1990), orthe signal described in WO 90/13646 published 15 Nov. 1990. In mammaliancell expression, mammalian signal sequences may be used to directsecretion of the protein, such as signal sequences from secretedpolypeptides of the same or related species, as well as viral secretoryleaders.

Both expression and cloning vectors contain a nucleic acid sequence thatenables the vector to replicate in one or more selected host cells. Suchsequences are well known for a variety of bacteria, yeast, and viruses.The origin of replication from the plasmid pBR322 is suitable for mostGram-negative bacteria, the 2μ plasmid origin is suitable for yeast, andvarious viral origins (SV40, polyoma, adenovirus or BPV) are useful forcloning vectors in mammalian cells.

Expression and cloning vectors will typically contain a selection gene,also termed a selectable marker. Typical selection genes encode proteinsthat (a) confer resistance to antibiotics or other toxins, e.g.,ampicillin, neomycin, methotrexate, or tetracycline, (b) complementauxotrophic deficiencies, or (c) supply critical nutrients not availablefrom complex media, e.g., the gene encoding D-alanine racemase forBacilli.

An example of suitable selectable markers for mammalian cells are thosethat enable the identification of cells competent to take up theimmunoadhesin-encoding nucleic acid, such as DHFR or thymidine kinase.An appropriate host cell when wild-type DHFR is employed is the CHO cellline deficient in DHFR activity, prepared and propagated as described byUrlaub et al., Proc. Natl. Acad. Sci. USA, 77:4216 (1980). A suitableselection gene for use in yeast is the trp1 gene present in the yeastplasmid YRp7 [Stinchcomb et al., Nature, 282:39 (1979); Kingsman et al.,Gene, 7:141 (1979); Tschemper et al., Gene, 10:157 (1980)]. The trp1gene provides a selection marker for a mutant strain of yeast lackingthe ability to grow in tryptophan, for example, ATCC No. 44076 or PEP4-1[Jones, Genetics, 85:12 (1977)].

Expression and cloning vectors usually contain a promoter operablylinked to the immunoadhesin-encoding nucleic acid sequence to directmRNA synthesis. Promoters recognized by a variety of potential hostcells are well known. Promoters suitable for use with prokaryotic hostsinclude the β-lactamase and lactose promoter systems [Chang et al.,Nature, 275:615 (1978); Goeddel et al., Nature, 281:544 (1979)],alkaline phosphatase, a tryptophan (trp) promoter system [Goeddel,Nucleic Acids Res., 8:4057 (1980); EP 36,776], and hybrid promoters suchas the tac promoter [deBoer et al., Proc. Natl. Acad. Sci. USA, 80:21-25(1983)]. Promoters for use in bacterial systems also will contain aShine-Dalgarno (S.D.) sequence operably linked to the DNA encodingimmunoadhesin.

Examples of suitable promoter sequences for use with yeast hosts includethe promoters for 3-phosphoglycerate kinase [Hitzeman et al., J. Biol.Chem., 255:2073 (1980)] or other glycolytic enzymes [Hess et al., J.Adv. Enzyme Reg., 7:149 (1968); Holland, Biochemistry, 17:4900 (1978)],such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase,pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphateisomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphateisomerase, phosphoglucose isomerase, and glucokinase.

Other yeast promoters, which are inducible promoters having theadditional advantage of transcription controlled by growth conditions,are the promoter regions for alcohol dehydrogenase 2, isocytochrome C,acid phosphatase, degradative enzymes associated with nitrogenmetabolism, metallothionein, glyceraldehyde-3-phosphate dehydrogenase,and enzymes responsible for maltose and galactose utilization. Suitablevectors and promoters for use in yeast expression are further describedin EP 73,657.

The transcription of immunoadhesin from vectors in mammalian host cellsis controlled, for example, by promoters obtained from the genomes ofviruses such as polyoma virus, fowlpox virus (UK 2,211,504 published 5Jul. 1989), adenovirus (such as Adenovirus 2), bovine papilloma virus,retrovirus (such as avian sarcoma virus), cytomegalovirus, hepatitis-Bvirus and Simian Virus 40 (SV40); from heterologous mammalian promoters,e.g., the actin promoter or an immunoglobulin promoter, or fromheat-shock promoters, provided such promoters are compatible with thehost cell systems.

Transcription of a DNA encoding the immunoadhesin by higher eukaryotesmay be increased by inserting an enhancer sequence into the vector.Enhancers are cis-acting elements of DNA, usually about from 10 to 300bp, that act on a promoter to increase its transcription. Many enhancersequences are now known from mammalian genes (globin, elastase, albumin,α-fetoprotein, and insulin). Typically, however, one will use anenhancer from a eukaryotic cell virus. Examples include the SV40enhancer on the late side of the replication origin (by 100-270), thecytomegalovirus early promoter enhancer, the polyoma enhancer on thelate side of the replication origin, and adenovirus enhancers. Theenhancer may be spliced into the vector at a position 5′ or 3′ to theimmunoadhesin coding sequence, but is preferably located at a site 5′from the promoter.

Expression vectors used in eukaryotic host cells (yeast, fungi, insect,plant, animal, human, or nucleated cells from other multicellularorganisms) will also contain sequences necessary for the termination oftranscription and for stabilizing the mRNA. Such sequences are commonlyavailable from the 3′ untranslated regions of eukaryotic or viral DNAsor cDNAs. These regions contain nucleotide segments transcribed aspolyadenylated fragments in the untranslated portion of the mRNAencoding immunoadhesin.

Still other methods, vectors, and host cells suitable for adaptation tothe synthesis of immunoadhesin in recombinant vertebrate cell cultureare described in Gething et al., Nature, 293:620-625 (1981); Mantei etal., Nature, 281:40-46 (1979); EP 117,060; and EP 117,058.

(iv) Purification of Immunoadhesin

An immunoadhesin or a chimeric heteroadhesin preferably is recoveredfrom the culture medium as a secreted polypeptide, although it also maybe recovered from host cell lysates. As a first step, the particulatedebris, either host cells or lysed fragments, is removed, for example,by centrifugation or ultrafiltration; optionally, the protein may beconcentrated with a commercially available protein concentration filter,followed by separating the chimeric heteroadhesin from other impuritiesby one or more purification procedures selected from: fractionation onan immunoaffinity column; fractionation on an ion-exchange column;ammonium sulphate or ethanol precipitation; reverse phase HPLC;chromatography on silica; chromatography on heparin Sepharose;chromatography on a cation exchange resin; chromatofocusing; SDS-PAGE;and gel filtration.

A particularly advantageous method of purifying immunoadhesins isaffinity chromatography. The choice of affinity ligand depends on thespecies and isotype of the immunoglobulin Fc domain that is used in thechimera. Protein A can be used to purify immunoadhesins that are basedon human IgG1, IgG2, or IgG4 heavy chains [Lindmark et al., J. Immunol.Meth. 62, 1-13 (1983)]. Protein G is recommended for all mouse isotypesand for human IgG3 [Guss et al., EMBO J. 5, 15671575 (1986)]. The matrixto which the affinity ligand is attached is most often agarose, butother matrices are also available. Mechanically stable matrices such ascontrolled pore glass or poly(styrenedivinyl)benzene allow for fasterflow rates and shorter processing times than can be achieved withagarose. The conditions for binding an immunoadhesin to the protein A orG affinity column are dictated entirely by the characteristics of the Fcdomain; that is, its species and isotype. Generally, when the properligand is chosen, efficient binding occurs directly from unconditionedculture fluid. One distinguishing feature of immunoadhesins is that, forhuman IgG1 molecules, the binding capacity for protein A is somewhatdiminished relative to an antibody of the same Fc type. Boundimmunoadhesin can be efficiently eluted either at acidic pH (at or above3.0), or in a neutral pH buffer containing a mildly chaotropic salt.This affinity chromatography step can result in an immunoadhesinpreparation that is >95% pure.

Other methods known in the art can be used in place of, or in additionto, affinity chromatography on protein A or G to purify immunoadhesins.Immunoadhesins behave similarly to antibodies in thiophilic gelchromatography [Hutchens and Porath, Anal. Biochem. 159, 217-226 (1986)]and immobilized metal chelate chromatography [Al-Mashikhi and Makai, J.Dairy Sci. 71, 1756-1763 (1988)]. In contrast to antibodies, however,their behavior on ion exchange columns is dictated not only by theirisoelectric points, but also by a charge dipole that may exist in themolecules due to their chimeric nature.

Preparation of epitope tagged immunoadhesin, such as ErbB4-IgG,facilitates purification using an immunoaffinity column containingantibody to the epitope to adsorb the fusion polypeptide. Immunoaffinitycolumns such as a rabbit polyclonal anti-ErbB4 column can be employed toabsorb the ErbB4-IgG by binding it to an ErbB4 immune epitope.

In some embodiments, the ErbB4 receptor-immunoglobulin chimeras(immunoadhesins) are assembled as monomers, or hetero- orhomo-multimers, and particularly as dimers or tetramers, essentially asillustrated in WO 91/08298. Generally, these assembled immunoglobulinswill have known unit structures. A basic four chain structural unit isthe form in which IgG, IgD, and IgE exist. A four-unit structure isrepeated in the higher molecular weight immunoglobulins; IgM generallyexists as a pentamer of basic four units held together by disulfidebonds. IgA globulin, and occasionally IgG globulin, may also exist inmultimeric form in serum. In the case of multimer, each four unit may bethe same or different.

As noted earlier, the immunoadhesins of the present invention can bemade bispecific, and may, for example, include binding regions from twodifferent ErbB receptors, at least one or which is ErbB4. Thus, theimmunoadhesins of the present invention may have binding specificitiesfor two distinct ErbB ligands. For bispecific molecules, trimericmolecules, composed of a chimeric antibody heavy chain in one arm and achimeric antibody heavy chain-light chain pair in the other arm of theirantibody-like structure are advantageous, due to ease of purification.In contrast to antibody-producing quadromas traditionally used for theproduction of bispecific immunoadhesins, which produce a mixture of tentetramers, cells transfected with nucleic acid encoding the three chainsof a trimeric immunoadhesin structure produce a mixture of only threemolecules, and purification of the desired product from this mixture iscorrespondingly easier.

(v) Characterization of Immunoadhesin

Generally, the ErbB4 chimeric heteromultimers of the invention will haveany one or more of the following properties: (a) the ability to competewith a natural heteromultimeric receptor for binding to a ligand such asHB-EGF; (b) the ability to form ErbB2-IgG/ErbB4-IgG complexes; and (c)the ability to inhibit activation of a natural heteromultimeric receptorby depleting ligand from the environment of the natural receptor,thereby inhibiting proliferation of cells that express the ErbB2 andErbB4 receptor.

To screen for property (a), the ability of the chimeric ErbB4heteromultimer adhesin to bind to a ligand can be readily determined invitro. For example, immunoadhesin forms of these receptors can begenerated and the ErbB2/4-Ig heteroimmunoadhesin can be immobilized on asolid phase (e.g. on assay plates coated with goat-anti-human antibody).The ability of a ligand to bind to the immobilized immunoadhesin canthen be determined. For more details, see the ¹²⁵I-HRG binding assaydescribed in the Example below.

As to property (c), the tyrosine phosphorylation assay using MCF7 cellsprovides a means for screening for activation of ErbB4 receptors. In analternative embodiment of the invention, the KIRA-ELISA described in WO95/14930 can be used to qualitatively and quantitatively measure theability of an HER4 chimeric heteroadhesin to inhibit activation of aHER4 receptor.

The ability of an immunoadhesin, chimeric heteroadhesin such asErbB2/4-Ig, or other molecule of the present invention to inhibitproliferation of a cell that expresses the ErbB2 and ErbB4 receptor isreadily determined in cell culture by standard procedures. Useful cellsfor this experiment include MCF7 and SK-BR-3 cells obtainable from theATCC and Schwann cells (see, for example, Li et al., J. Neuroscience16(6):2012-2019 (1996)). These tumor cell lines may be plated in cellculture plates and allowed to adhere thereto. The HRG ligand in thepresence and absence of a potential ErbB4 antagonist such as an ErbB4chimeric heteroadhesin is added. Monolayers are washed and stained/fixedwith crystal violet and cell growth inhibition is quantified.

2. Antibody Preparation

Another preferred class of ErbB4 antagonists comprises neutralizingantibodies to this receptor.

(i) Polyclonal Antibodies

Methods of preparing polyclonal antibodies are known in the art.Polyclonal antibodies can be raised in a mammal, for example, by one ormore injections of an immunizing agent and, if desired, an adjuvant.Typically, the immunizing agent and/or adjuvant will be injected in themammal by multiple subcutaneous or intraperitoneal injections. It may beuseful to conjugate the immunizing agent to a protein known to beimmunogenic in the mammal being immunized, such as serum albumin, orsoybean trypsin inhibitor. Examples of adjuvants which may be employedinclude Freund's complete adjuvant and MPL-TDM.

(ii) Monoclonal Antibodies

Monoclonal antibodies may be made using the hybridoma method firstdescribed by Kohler et al., Nature, 256:495 (1975), or may be made byrecombinant DNA methods (U.S. Pat. No. 4,816,567).

In the hybridoma method, a mouse or other appropriate host animal, suchas a hamster or macaque monkey, is immunized as hereinabove described toelicit lymphocytes that produce or are capable of producing antibodiesthat will specifically bind to the protein used for immunization.Alternatively, lymphocytes may be immunized in vitro. Lymphocytes thenare fused with myeloma cells using a suitable fusing agent, such aspolyethylene glycol, to form a hybridoma cell (Goding, MonoclonalAntibodies. Principles and Practice, pp. 59-103, [Academic Press,1986]).

The hybridoma cells thus prepared are seeded and grown in a suitableculture medium that preferably contains one or more substances thatinhibit the growth or survival of the unfused, parental myeloma cells.For example, if the parental myeloma cells lack the enzyme hypoxanthineguanine phosphoribosyl transferase (HGPRT or HPRT), the culture mediumfor the hybridomas typically will include hypoxanthine, aminopterin, andthymidine (HAT medium), which substances prevent the growth ofHGPRT-deficient cells.

Preferred myeloma cells are those that fuse efficiently, support stablehigh-level production of antibody by the selected antibody-producingcells, and are sensitive to a medium such as HAT medium. Among these,preferred myeloma cell lines are murine myeloma lines, such as thosederived from MOP-21 and MC.-11 mouse tumors available from the SalkInstitute Cell Distribution Center, San Diego, Calif. USA, and SP-2 orX63-Ag8-653 cells available from the American Type Culture Collection,Rockville, Md. USA. Human myeloma and mouse-human heteromyeloma celllines also have been described for the production of human monoclonalantibodies (Kozbor, J. Immunol., 133:3001 (1984); Brodeur et al.,Monoclonal Antibody Production Techniques and Applications, pp. 51-63,Marcel Dekker, Inc., New York, [1987]).

Culture medium in which hybridoma cells are growing is assayed forproduction of monoclonal antibodies directed against the antigen.Preferably, the binding specificity of monoclonal antibodies produced byhybridoma cells is determined by immunoprecipitation or by an in vitrobinding assay, such as radioimmunoassay (RIA) or enzyme-linkedimmunosorbent assay (ELISA).

The binding affinity of the monoclonal antibody can, for example, bedetermined by the Scatchard analysis of Munson et al., Anal. Biochem.,107:220 (1980).

After hybridoma cells are identified that produce antibodies of thedesired specificity, affinity, and/or activity, the cells may besubcloned by limiting dilution procedures and grown by standard methods(Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103(Academic Press, 1986)). Suitable culture media for this purposeinclude, for example, DMEM or RPMI-1640 medium. In addition, thehybridoma cells may be grown in vivo as ascites tumors in an animal.

The monoclonal antibodies secreted by the subclones are suitablyseparated from the culture medium, ascites fluid, or serum byconventional immunoglobulin purification procedures such as, forexample, protein A-Sepharose, hydroxylapatite chromatography, gelelectrophoresis, dialysis, or affinity chromatography.

DNA encoding the monoclonal antibodies is readily isolated and sequencedusing conventional procedures (e.g., by using oligonucleotide probesthat are capable of binding specifically to genes encoding the heavy andlight chains of the monoclonal antibodies). The hybridoma cells serve asa preferred source of such DNA. Once isolated, the DNA may be placedinto expression vectors, which are then transfected into host cells suchas E. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells,or myeloma cells that do not otherwise produce immunoglobulin protein,to obtain the synthesis of monoclonal antibodies in the recombinant hostcells. The DNA also may be modified, for example, by substituting thecoding sequence for human heavy and light chain constant domains inplace of the homologous murine sequences, Morrison, et al., Proc. Nat.Acad. Sci. 81, 6851 (1984), or by covalently joining to theimmunoglobulin coding sequence all or part of the coding sequence for anon-immunoglobulin polypeptide. In that manner, “chimeric” or “hybrid”antibodies are prepared that have the binding specificity of ananti-ErbB4 receptor monoclonal antibody herein.

Typically such non-immunoglobulin polypeptides are substituted for theconstant domains of an antibody of the invention, or they aresubstituted for the variable domains of one antigen-combining site of anantibody of the invention to create a chimeric bivalent antibodycomprising one antigen-combining site having specificity for a ErbB4receptor and another antigen-combining site having specificity for adifferent antigen.

Chimeric or hybrid antibodies also may be prepared in vitro using knownmethods in synthetic protein chemistry, including those involvingcrosslinking agents. For example, immunotoxins may be constructed usinga disulfide exchange reaction or by forming a thioether bond. Examplesof suitable reagents for this purpose include iminothiolate andmethyl-4-mercaptobutyrimidate.

Recombinant production of antibodies will be described in more detailbelow.

(iii) Humanized Antibodies

Generally, a humanized antibody has one or more amino acid residuesintroduced into it from a non-human source. These non-human amino acidresidues are often referred to as “import” residues, which are typicallytaken from an “import” variable domain. Humanization can be essentiallyperformed following the method of Winter and co-workers [Jones et al.,Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-327(1988); Verhoeyen et al., Science, 239:1534-1536 (1988)], bysubstituting rodent CDRs or CDR sequences for the correspondingsequences of a human antibody.

Accordingly, such “humanized” antibodies are chimeric antibodies(Cabilly, supra), wherein substantially less than an intact humanvariable domain has been substituted by the corresponding sequence froma non-human species. In practice, humanized antibodies are typicallyhuman antibodies in which some CDR residues and possibly some FRresidues are substituted by residues from analogous sites in rodentantibodies.

It is important that antibodies be humanized with retention of highaffinity for the antigen and other favorable biological properties. Toachieve this goal, according to a preferred method, humanized antibodiesare prepared by a process of analysis of the parental sequences andvarious conceptual humanized products using three-dimensional models ofthe parental and humanized sequences. Three dimensional immunoglobulinmodels are commonly available and are familiar to those skilled in theart. Computer programs are available which illustrate and displayprobable three-dimensional conformational structures of selectedcandidate immunoglobulin sequences. Inspection of these displays permitsanalysis of the likely role of the residues in the functioning of thecandidate immunoglobulin sequence, i.e. the analysis of residues thatinfluence the ability of the candidate immunoglobulin to bind itsantigen. In this way, FR residues can be selected and combined from theconsensus and import sequence so that the desired antibodycharacteristic, such as increased affinity for the target antigen(s), isachieved. In general, the CDR residues are directly and mostsubstantially involved in influencing antigen binding. For furtherdetails, see U.S. Pat. No. 5,821,337.

(iv) Human Antibodies

Human monoclonal antibodies can be made by the hybridoma method. Humanmyeloma and mouse-human heteromyeloma cell lines for the production ofhuman monoclonal antibodies have been described, for example, by Kozbor,J. Immunol. 133, 3001 (1984), and Brodeur, et al., Monoclonal AntibodyProduction Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc.,New York, 1987).

It is now possible to produce transgenic animals (e.g. mice) that arecapable, upon immunization, of producing a repertoire of humanantibodies in the absence of endogenous immunoglobulin production. Forexample, it has been described that the homozygous deletion of theantibody heavy chain joining region (J_(H)) gene in chimeric andgerm-line mutant mice results in complete inhibition of endogenousantibody production. Transfer of the human germ-line immunoglobulin genearray in such germ-line mutant mice will result in the production ofhuman antibodies upon antigen challenge. See, e.g. Jakobovits et al.,Proc. Natl. Acad. Sci. USA 90, 2551-255 (1993); Jakobovits et al.,Nature 362, 255-258 (1993).

Mendez et al. (Nature Genetics 15: 146-156 [1997]) have further improvedthe technology and have generated a line of transgenic mice designatedas “Xenomouse II” that, when challenged with an antigen, generates highaffinity fully human antibodies. This was achieved by germ-lineintegration of megabase human heavy chain and light chain loci into micewith deletion into endogenous J_(H) segment as described above. TheXenomouse II harbors 1,020 kb of human heavy chain locus containingapproximately 66 V_(H) genes, complete D_(H) and J_(H) regions and threedifferent constant regions (μ, δ and χ), and also harbors 800 kb ofhuman κ locus containing 32 Vκ genes, Jκ segments and Cκ genes. Theantibodies produced in these mice closely resemble that seen in humansin all respects, including gene rearrangement, assembly, and repertoire.The human antibodies are preferentially expressed over endogenousantibodies due to deletion in endogenous J_(H) segment that preventsgene rearrangement in the murine locus.

Alternatively, the phage display technology (McCafferty et al., Nature348, 552-553 [1990]) can be used to produce human antibodies andantibody fragments in vitro, from immunoglobulin variable (V) domaingene repertoires from unimmunized donors. According to this technique,antibody V domain genes are cloned in-frame into either a major or minorcoat protein gene of a filamentous bacteriophage, such as M13 or fd, anddisplayed as functional antibody fragments on the surface of the phageparticle. Because the filamentous particle contains a single-strandedDNA copy of the phage genome, selections based on the functionalproperties of the antibody also result in selection of the gene encodingthe antibody exhibiting those properties. Thus, the phage mimics some ofthe properties of the B-cell. Phage display can be performed in avariety of formats; for their review see, e.g. Johnson, Kevin S. andChiswell, David J., Current Opinion in Structural Biology 3, 564-571(1993). Several sources of V-gene segments can be used for phagedisplay. Clackson et al., Nature 352, 624-628 (1991) isolated a diversearray of anti-oxazolone antibodies from a small random combinatoriallibrary of V genes derived from the spleens of immunized mice. Arepertoire of V genes from unimmunized human donors can be constructedand antibodies to a diverse array of antigens (including self-antigens)can be isolated essentially following the techniques described by Markset al., J. Mol. Biol. 222, 581-597 (1991), or Griffiths et al., EMBO J.12, 725-734 (1993). In a natural immune response, antibody genesaccumulate mutations at a high rate (somatic hypermutation). Some of thechanges introduced will confer higher affinity, and B cells displayinghigh-affinity surface immunoglobulin are preferentially replicated anddifferentiated during subsequent antigen challenge. This natural processcan be mimicked by employing the technique known as “chain shuffling”(Marks et al., Bio/Technol. 10, 779-783 [1992]). In this method, theaffinity of “primary” human antibodies obtained by phage display can beimproved by sequentially replacing the heavy and light chain V regiongenes with repertoires of naturally occurring variants (repertoires) ofV domain genes obtained from unimmunized donors. This techniques allowsthe production of antibodies and antibody fragments with affinities inthe nM range. A strategy for making very large phage antibodyrepertoires (also known as “the mother-of-all libraries”) has beendescribed by Waterhouse et al., Nucl. Acids Res. 21, 2265-2266 (1993),and the isolation of a high affinity human antibody directly from suchlarge phage library is reported by Griffiths et al., EMBO J. 13:3245-3260 (1994). Gene shuffling can also be used to derive humanantibodies from rodent antibodies, where the human antibody has similaraffinities and specificities to the starting rodent antibody. Accordingto this method, which is also referred to as “epitope imprinting”, theheavy or light chain V domain gene of rodent antibodies obtained byphage display technique is replaced with a repertoire of human V domaingenes, creating rodent-human chimeras. Selection on antigen results inisolation of human variable domains capable of restoring a functionalantigen-binding site, i.e. the epitope governs (imprints) the choice ofpartner. When the process is repeated in order to replace the remainingrodent V domain, a human antibody is obtained (see PCT patentapplication WO 93/06213, published 1 Apr. 1993). Unlike traditionalhumanization of rodent antibodies by CDR grafting, this techniqueprovides completely human antibodies, which have no framework or CDRresidues of rodent origin.

(v) Bispecific Antibodies

Bispecific antibodies are monoclonal, preferably human or humanized,antibodies that have binding specificities for at least two differentantigens. In the present case, one of the binding specificities is forthe ErbB4 receptor to provide an antagonist antibody, the other one isfor any other antigen, and preferably for another receptor or receptorsubunit.

Methods for making bispecific antibodies are known in the art.Traditionally, the recombinant production of bispecific antibodies isbased on the co-expression of two immunoglobulin heavy chain-light chainpairs, where the two heavy chains have different specificities(Millstein and Cuello, Nature 305, 537-539 (1983)). Because of therandom assortment of immunoglobulin heavy and light chains, thesehybridomas (quadromas) produce a potential mixture of 10 differentantibody molecules, of which only one has the correct bispecificstructure. The purification of the correct molecule, which is usuallydone by affinity chromatography steps, is rather cumbersome, and theproduct yields are low. Similar procedures are disclosed in PCTapplication publication No. WO 93/08829 (published 13 May 1993), and inTraunecker et al., EMBO 10, 3655-3659 (1991).

According to a different and more preferred approach, antibody variabledomains with the desired binding specificities (antibody-antigencombining sites) are fused to immunoglobulin constant domain sequences.The fusion preferably is with an immunoglobulin heavy chain constantdomain, comprising at least part of the hinge, CH2 and CH3 regions. Itis preferred to have the first heavy chain constant region (CH1)containing the site necessary for light chain binding, present in atleast one of the fusions. DNAs encoding the immunoglobulin heavy chainfusions and, if desired, the immunoglobulin light chain, are insertedinto separate expression vectors, and are co-transfected into a suitablehost organism. This provides for great flexibility in adjusting themutual proportions of the three polypeptide fragments in embodimentswhen unequal ratios of the three polypeptide chains used in theconstruction provide the optimum yields. It is, however, possible toinsert the coding sequences for two or all three polypeptide chains inone expression vector when the expression of at least two polypeptidechains in equal ratios results in high yields or when the ratios are ofno particular significance. In a preferred embodiment of this approach,the bispecific antibodies are composed of a hybrid immunoglobulin heavychain with a first binding specificity in one arm, and a hybridimmunoglobulin heavy chain-light chain pair (providing a second bindingspecificity) in the other arm. It was found that this asymmetricstructure facilitates the separation of the desired bispecific compoundfrom unwanted immunoglobulin chain combinations, as the presence of animmunoglobulin light chain in only one half of the bispecific moleculeprovides for a facile way of separation.

For further details of generating bispecific antibodies see, forexample, Suresh et al., Methods in Enzymology 121, 210 (1986).

(vi) Heteroconjugate Antibodies

Heteroconjugate antibodies are also within the scope of the presentinvention. Heteroconjugate antibodies are composed of two covalentlyjoined antibodies. Such antibodies have, for example, been proposed totarget immune system cells to unwanted cells (U.S. Pat. No. 4,676,980),and for treatment of HIV infection (PCT application publication Nos. WO91/00360 and WO 92/200373; EP 03089). Heteroconjugate antibodies may bemade using any convenient cross-linking methods. Suitable cross-linkingagents are well known in the art, and are disclosed in U.S. Pat. No.4,676,980, along with a number of cross-linking techniques.

(vii) Antibody Fragments

In certain embodiments, the ErbB4 antagonist antibody (including murine,human and humanized antibodies, and antibody variants) is an antibodyfragment. Various techniques have been developed for the production ofantibody fragments. Traditionally, these fragments were derived viaproteolytic digestion of intact antibodies (see, e.g., Morimoto et al.,J. Biochem. Biophys. Methods 24:107-117 (1992) and Brennan et al.,Science 229:81 (1985)). However, these fragments can now be produceddirectly by recombinant host cells. For example, Fab′-SH fragments canbe directly recovered from E. coli and chemically coupled to formF(ab′)₂ fragments (Carter et al., Bio/Technology 10:163-167 (1992)). Inanother embodiment, the F(ab′)₂ is formed using the leucine zipper GCN4to promote assembly of the F(ab′)₂ molecule. According to anotherapproach, Fv, Fab or F(ab′)₂ fragments can be isolated directly fromrecombinant host cell culture. Other techniques for the production ofantibody fragments will be apparent to the skilled practitioner.

(viii) Amino Acid Sequence Variants of Antibodies

Amino acid sequence variants of the ErbB4 antagonist antibodies areprepared by introducing appropriate nucleotide changes into the ErbB4antagonist antibody DNA, or by peptide synthesis. Such variants include,for example, deletions from, and/or insertions into and/or substitutionsof, residues within the amino acid sequences of the ErbB4 antagonistantibodies of the examples shown herein. Any combination of deletion,insertion, and substitution is made to arrive at the final construct,provided that the final construct possesses the desired characteristics.The amino acid changes also may alter post-translational processes ofthe humanized or variant ErbB4 antagonist antibody, such as changing thenumber or position of glycosylation sites.

A useful method for identification of certain residues or regions of theErbB4 receptor antibody that are preferred locations for mutagenesis iscalled “alanine scanning mutagenesis,” as described by Cunningham andWells Science, 244:1081-1085 (1989). Here, a residue or group of targetresidues are identified (e.g., charged residues such as arg, asp, his,lys, and glu) and replaced by a neutral or negatively charged amino acid(most preferably alanine or polyalanine) to affect the interaction ofthe amino acids with ErbB4 receptor antigen. Those amino acid locationsdemonstrating functional sensitivity to the substitutions then arerefined by introducing further or other variants at, or for, the sitesof substitution. Thus, while the site for introducing an amino acidsequence variation is predetermined, the nature of the mutation per seneed not be predetermined. For example, to analyze the performance of amutation at a given site, ala scanning or random mutagenesis isconducted at the target codon or region and the expressed ErbB4 antibodyvariants are screened for the desired activity.

Amino acid sequence insertions include amino- and/or carboxyl-terminalfusions ranging in length from one residue to polypeptides containing ahundred or more residues, as well as intrasequence insertions of singleor multiple amino acid residues. Examples of terminal insertions includea ErbB4 antagonist antibody with an N-terminal methionyl residue or theantibody fused to an epitope tag. Other insertional variants of theErbB4 antagonist antibody molecule include the fusion to the N- orC-terminus of the ErbB4 antagonist antibody of an enzyme or apolypeptide which increases the serum half-life of the antibody.

Another type of variant is an amino acid substitution variant. Thesevariants have at least one amino acid residue in the ErbB4 antagonistantibody molecule removed and a different residue inserted in its place.The sites of greatest interest for substitution mutagenesis include thehypervariable regions, but FR alterations are also contemplated.Conservative substitutions are shown in Table 1 under the heading of“preferred substitutions”. If such substitutions result in a change inbiological activity, then more substantial changes, denominated“exemplary substitutions” in Table 1, or as further described below inreference to amino acid classes, may be introduced and the productsscreened.

TABLE 1 Exemplary Preferred Original Residue Substitutions SubstitutionsAla (A) val; leu; ile val Arg (R) lys, gln, asn lys Asn (N) gln; his;asp, gln lys; arg Asp (D) glu; asn glu Cys (C) ser; ala ser Gln (Q) asn;glu asn Glu (E) asp; gln asp Gly (G) ala ala His (H) asn; gln; arg lys;arg Ile (I) leu; val; met; leu ala; phe; norleucine Leu (L) norleucine;ile; ile val; met; ala; phe Lys (K) arg; gln; asn arg Met (M) leu; phe;ile leu Phe (F) leu; val; ile; tyr ala; tyr Pro (P) ala ala Ser (S) thrthr Thr (T) ser ser Trp (W) tyr; phe tyr Tyr (Y) trp; phe; phe thr; serVal (V) ile; leu; met; leu phe; ala; norleucine

Substantial modifications in the biological properties of the antibodyare accomplished by selecting substitutions that differ significantly intheir effect on maintaining (a) the structure of the polypeptidebackbone in the area of the substitution, for example, as a sheet orhelical conformation, (b) the charge or hydrophobicity of the moleculeat the target site, or (c) the bulk of the side chain. Naturallyoccurring residues are divided into groups based on common side-chainproperties:

(1) hydrophobic: norleucine, met, ala, val, leu, ile;

(2) neutral hydrophilic: cys, ser, thr;

(3) acidic: asp, glu;

(4) basic: asn, gin, his, lys, arg;

(5) residues that influence chain orientation: gly, pro; and

(6) aromatic: trp, tyr, phe.

Non-conservative substitutions will entail exchanging a member of one ofthese classes for another class.

Any cysteine residue not involved in maintaining the proper conformationof the ErbB4 antagonist antibody also may be substituted, generally withserine, to improve the oxidative stability of the molecule and preventaberrant crosslinking. Conversely, cysteine bond(s) may be added to theantibody to improve its stability (particularly where the antibody is anantibody fragment such as a Fv fragment).

A particularly preferred type of substitution variant involvessubstituting one or more hypervariable region residues of a parentantibody (e.g. a humanized or human antibody). Generally, the resultingvariant(s) selected for further development will have improvedbiological properties relative to the parent antibody from which theyare generated. A convenient way for generating such substitutionvariants is affinity maturation using phage display. Briefly, severalhypervariable region sites (e.g. 6-7 sites) are mutated to generate allpossible amino substitutions at each site. The antibody variants thusgenerated are displayed in a monovalent fashion from filamentous phageparticles as fusions to the gene III product of M13 packaged within eachparticle. The phage-displayed variants are then screened for theirbiological activity (e.g. antagonist activity) as herein disclosed. Inorder to identify candidate hypervariable region sites for modification,alanine scanning mutagenesis can be performed to identify hypervariableregion residues contributing significantly to antigen binding.Alternatively, or in addition, it may be beneficial to analyze a crystalstructure of the antigen-antibody complex to identify contact pointsbetween the antibody and ErbB receptor. Such contact residues andneighboring residues are candidates for substitution according to thetechniques elaborated herein. Once such variants are generated, thepanel of variants is subjected to screening as described herein andantibodies with superior properties in one or more relevant assays maybe selected for further development.

Another type of amino acid variant of the antibody alters the originalglycosylation pattern of the antibody. By altering is meant deleting oneor more carbohydrate moieties found in the antibody, and/or adding oneor more glycosylation sites that are not present in the antibody.

Glycosylation of antibodies is typically either N-linked or O-linked.N-linked refers to the attachment of the carbohydrate moiety to the sidechain of an asparagine residue. The tripeptide sequencesasparagine-X-serine and asparagine-X-threonine, where X is any aminoacid except proline, are the recognition sequences for enzymaticattachment of the carbohydrate moiety to the asparagine side chain.Thus, the presence of either of these tripeptide sequences in apolypeptide creates a potential glycosylation site. O-linkedglycosylation refers to the attachment of one of the sugarsN-aceylgalactosamine, galactose, or xylose to a hydroxyamino acid, mostcommonly serine or threonine, although 5-hydroxyproline or5-hydroxylysine may also be used.

Addition of glycosylation sites to the antibody is convenientlyaccomplished by altering the amino acid sequence such that it containsone or more of the above-described tripeptide sequences (for N-linkedglycosylation sites). The alteration may also be made by the additionof, or substitution by, one or more serine or threonine residues to thesequence of the original antibody (for O-linked glycosylation sites).

Nucleic acid molecules encoding amino acid sequence variants of theErbB4 antagonist antibodies are prepared by a variety of methods knownin the art. These methods include, but are not limited to, isolationfrom a natural source (in the case of naturally occurring amino acidsequence variants) or preparation by oligonucleotide-mediated (orsite-directed) mutagenesis, PCR mutagenesis, and cassette mutagenesis ofan earlier prepared variant or a non-variant version of the ErbB4antagonist antibody.

(ix) Other Modifications of Antibodies

The ErbB4 antagonist antibodies disclosed herein may also be formulatedas immunoliposomes. Liposomes containing the antibody are prepared bymethods known in the art, such as described in Epstein et al., Proc.Natl. Acad. Sci. USA 82:3688 (1985); Hwang et al., Proc. Natl. Acad.Sci. USA 77:4030 (1980); and U.S. Pat. Nos. 4,485,045 and 4,544,545.Liposomes with enhanced circulation time are disclosed in U.S. Pat. No.5,013,556.

Particularly useful liposomes can be generated by the reverse phaseevaporation method with a lipid composition comprisingphosphatidylcholine, cholesterol and PEG-derivatizedphosphatidylethanolamine (PEG-PE). Liposomes are extruded throughfilters of defined pore size to yield liposomes with the desireddiameter. Fab′ fragments of the antibody of the present invention can beconjugated to the liposomes as described in Martin et al., J. Biol.Chem. 257:286-288 (1982) via a disulfide interchange reaction. Achemotherapeutic agent (such as Doxorubicin) is optionally containedwithin the liposome. See Gabizon et al., J. National Cancer Inst.81(19):1484 (1989).

The antibody of the present invention may also be used in ADEPT byconjugating the antibody to a prodrug-activating enzyme which converts aprodrug (e.g., a peptidyl chemotherapeutic agent, see WO81/01145) to anactive anti-cancer drug. See, for example, WO 88/07378 and U.S. Pat. No.4,975,278.

The enzyme component of the immunoconjugate useful for ADEPT includesany enzyme capable of acting on a prodrug in such a way so as to covertit into its more active, cytotoxic form.

Enzymes that are useful in the method of this invention include, but arenot limited to, alkaline phosphatase useful for convertingphosphate-containing prodrugs into free drugs; arylsulfatase useful forconverting sulfate-containing prodrugs into free drugs; cytosinedeaminase useful for converting non-toxic 5-fluorocytosine into theanti-cancer drug, 5-fluorouracil; proteases, such as serratia protease,thermolysin, subtilisin, carboxypeptidases and cathepsins (such ascathepsins B and L), that are useful for converting peptide-containingprodrugs into free drugs; D-alanylcarboxypeptidases, useful forconverting prodrugs that contain D-amino acid substituents;carbohydrate-cleaving enzymes such as β-galactosidase and neuraminidaseuseful for converting glycosylated prodrugs into free drugs; β-lactamaseuseful for converting drugs derivatized with β-lactams into free drugs;and penicillin amidases, such as penicillin V amidase or penicillin Gamidase, useful for converting drugs derivatized at their aminenitrogens with phenoxyacetyl or phenylacetyl groups, respectively, intofree drugs. Alternatively, antibodies with enzymatic activity, alsoknown in the art as “abzymes”, can be used to convert the prodrugs ofthe invention into free active drugs (see, e.g., Massey, Nature328:457-458 (1987)). Antibody-abzyme conjugates can be prepared asdescribed herein for delivery of the abzyme to a tumor cell population.

The enzymes of this invention can be covalently bound to the ErbB4antagonist antibodies by techniques well known in the art such as theuse of the heterobifunctional crosslinking reagents discussed above.Alternatively, fusion proteins comprising at least the antigen bindingregion of an antibody of the invention linked to at least a functionallyactive portion of an enzyme of the invention can be constructed usingrecombinant DNA techniques well known in the art (see, e.g., Neubergeret al., Nature 312:604-608 [1984]).

In certain embodiments of the invention, it may be desirable to use anantibody fragment, rather than an intact antibody. In this case, it maybe desirable to modify the antibody fragment in order to increase itsserum half-life. This may be achieved, for example, by incorporation ofa salvage receptor binding epitope into the antibody fragment (e.g., bymutation of the appropriate region in the antibody fragment or byincorporating the epitope into a peptide tag that is then fused to theantibody fragment at either end or in the middle, e.g., by DNA orpeptide synthesis). See WO96/32478 published Oct. 17, 1996.

The salvage receptor binding epitope generally constitutes a regionwherein any one or more amino acid residues from one or two loops of aFc domain are transferred to an analogous position of the antibodyfragment. Even more preferably, three or more residues from one or twoloops of the Fc domain are transferred. Still more preferred, theepitope is taken from the CH2 domain of the Fc region (e.g., of an IgG)and transferred to the CH1, CH3, or V_(H) region, or more than one suchregion, of the antibody. Alternatively, the epitope is taken from theCH2 domain of the Fc region and transferred to the C_(L) region or V_(L)region, or both, of the antibody fragment.

Covalent modifications of the ErbB4 antagonist antibodies are alsoincluded within the scope of this invention. They may be made bychemical synthesis or by enzymatic or chemical cleavage of the antibody,if applicable. Other types of covalent modifications of the antibody areintroduced into the molecule by reacting targeted amino acid residues ofthe antibody with an organic derivatizing agent that is capable ofreacting with selected side chains or the N- or C-terminal residues.Exemplary covalent modifications of polypeptides are described in U.S.Pat. No. 5,534,615, specifically incorporated herein by reference. Apreferred type of covalent modification of the antibody compriseslinking the antibody to one of a variety of nonproteinaceous polymers,e.g., polyethylene glycol, polypropylene glycol, or polyoxyalkylenes, inthe manner set forth in U.S. Pat. No. 4,640,835; 4,496,689; 4,301,144;4,670,417; 4,791,192 or 4,179,337.

3. Preparation of Soluble ErbB4 Receptors

Soluble ErbB4 receptors, such an ErbB4 extracellular domain, can beprepared by culturing cells transformed or transfected with a vectorcontaining the encoding nucleic acid. It is, of course, contemplatedthat alternative methods, which are well known in the art, may beemployed to prepare such soluble receptors. For instance, the solublereceptor sequence, or portions thereof, may be produced by directpeptide synthesis using solid-phase techniques (see, e.g., Stewart etal., supra; and Merrifield, supra). In vitro protein synthesis may beperformed using manual techniques or by automation. Automated synthesismay be accomplished, for instance, using an Applied Biosystems PeptideSynthesizer (Foster City, Calif.) using manufacturer's instructions.Various portions of the soluble receptor may be chemically synthesizedseparately and combined using chemical or enzymatic methods to producethe full-length soluble receptor.

Recombinant production of soluble ErbB4 receptors is performedessentially as described hereinabove in connection with immunoadhesins.

The most convenient method for the large-scale production of solubleErbB4 receptors is by cleavage from an ErbB4-Ig immunoadhesin. Thestructural similarity between immunoadhesins and antibodies suggestedthat it might be possible to cleave immunoadhesins by proteolyticenzymes such as papain, to generate Fd-like fragments containing the“adhesin” portion. In order to provide a more generic approach forcleavage of immunoadhesins, proteases which are highly specific fortheir target sequence are to be used. A protease suitable for thispurpose is an engineered mutant of subtilisin BPN, which recognizes andcleaves the sequence AAHYTL. Introduction of this target sequence intothe support hinge region of an ErbB4-IgG (e.g. IgG1) immunoadhesinfacilitates highly specific cleavage between the Fc and trk domains. TheIgG1 immunoadhesin is purified by protein A chromatography and cleavedwith an immobilized form of the enzyme. Cleavage results in twoproducts; the Fc region and the ErbB4 region, which is preferably anErbB4 extracellular domain. These fragments can be separated easily by asecond passage over a protein A column to retain the Fc and obtain thepurified ErbB4 fragment in the flow-through fractions. A similarapproach can be used to generate a dimeric ErbB4 portion, by placing thecleavable sequence at the lower hinge.

4. Therapeutic Compositions and Methods

The members of the ErbB family of receptors and corresponding ligandsare involved in smooth muscle cell proliferation in various organs.Accordingly, an ErbB4 receptor antagonist may be utilized for thetreatment of a variety of “diseases or disorders” involving smoothmuscle cell proliferation in a mammal, such as a human.

In a preferred embodiment, the present invention concerns the use ofErbB4 receptor antagonists for the treatment of cardiac diseasesinvolving proliferation of vascular smooth muscle cells (VSMC) andleading to intimal hyperplasia such as vascular stenosis, restenosisresulting from angioplasty or surgery or stent implants, atherosclerosisand hypertension (reviewed in Casterella and Teirstein, Cardiol. Rev. 7:219-231 [1999]; Andres, Int. J. Mol. Med. 2: 81-89 [1998]; and Rosanioet al., Thromb. Haemost. 82 [suppl 1]: 164-170 [1999]). There is anintricate interplay of various cells and cytokines released that act inautocrine, paracrine or juxtacrine manner, which result in migration ofVSMCs from their normal location in media to the damaged intima. Themigrated VSMCs proliferate excessively and lead to thickening of intima,which results in stenosis or occlusion of blood vessels. The problem iscompounded by platelet aggregation and deposition at the site of lesion.α-thrombin, a multifunctional serine protease, is concentrated at siteof vascular injury and stimulates VSMCs proliferation. Followingactivation of this receptor, VSMCs produce and secrete various autocrinegrowth factors, including PDGF-AA, HB-EGF and TGF-β (reviewed inStouffer and Runge, Semin. Thromb. Hemost. 24: 145-150 [1998]).

Various members of the EGF family play important roles in the normalgrowth and maintenance of blood vessels as well as in pathologicalconditions. For example, heparin-binding EGF-like growth factor (HB-EGF)is a potent mitogen and a chemotactic factor for fibroblasts as well asVSMCs but not endothelial cells (reviewed in Raab and Klagsbrun,Biochim. Biophys. Acta 1333: F179-199 [1997]). Vascular endothelialgrowth factor (VEGF), a powerful angiogenic factor, induces theexpression of HB-EGF in vascular endothelial cells (Arkonac et al., J.Biol. Chem. 273: 4400-4405 [1998]). HB-EGF binds to and activates HER1and ErbB4 receptors initiating a signal transduction cascade thatultimately results in migration and proliferation of fibroblasts andVSMCs. HB-EGF also stimulates VSMCs to secrete various factors that aremitogenic for endothelial cells (Abramovitch et al., FEBS Lett. 425:441-447 [1998]). Moreover, it also induces chemotactic response inendothelial cells. Similarly, another ligand that activates EGFreceptors, epiregulin, is secreted by VSMCs stimulated with angiotensinII, endothelin-1 and thrombin, and also acts as a powerful mitogen forproliferation of VSMCs (Taylor et al., Proc. Natl. Acad. Sci. USA 96:1633-1638 [1999]).

Vascular stenosis gives rise to hypertension as a result of increasedresistance to blood flow. Moreover, decreased blood supply to the tissuemay also cause necrosis and induce inflammatory response leading tosevere damage. For example, myocardial infarction occurs as a result oflack of oxygen and local death of heart muscle tissues. Percutaneoustransluminal coronary angioplasty (PTCA), simply referred to as balloonangioplasty, is a non-surgical catheter-based treatment for obstructivecoronary artery disease. In this method, a catheter is introduced in theblood vessel and a balloon is inflated at the site of plaque in order tomechanically dislodge the plaque. Alternatively, stent is implanted torestore smooth blood flow. However, neointimal formation takes placeeven within the implanted stent, known as “in-stent restenosis.” Forexample, stent deployment results in early thrombus deposition and acuteinflammation, granulation tissue development, and ultimately smoothmuscle cell proliferation and extracellular matrix synthesis (reviewedin Virmani and Farb, Curr. Opin. Lipidol. 10: 499-506 [1999]). Bypasssurgery is performed to get around the affected blood vessel only insevere cases, and usually only after multiple rounds of angioplasty havefailed in restoring blood flow.

Although balloon angioplasty has been used widely for the treatment ofstenosis, its long-term success is limited by restenosis. Restenosispersists as the limiting factor in the maintenance of vessel patencyafter PTCA, occurring in 30-50% of patients and accounting forsignificant morbidity and health care expenditure. The underlyingmechanisms of restenosis are comprised of a combination of effects fromvessel recoil, negative vascular remodeling, thrombus formation andneointimal hyperplasia. Importantly, these events are interconnected.For example, neointimal hyperplasia is stimulated by growth factors,which are released by local thrombi and the injured arterial segmentitself, and act to enhance the expression of other growth-stimulatingproteins resulting in acute proliferative and inflammatory responses.For instance, endothelial injury induces expression of EGF, EGF-likefactors and EGFR in VSMCs, which act upon them in an autocrine manner tostimulate their proliferation leading to intimal thickening andrestenosis. Extracellular matrix (ECM) formation and accumulation in thevessel wall is another important component of the restenosis lesion thatdevelops after balloon angioplasty.

A multitude of pharmacological trials have been conducted in an attemptto prevent restenosis, but most have demonstrated little benefits. Earlyclinical trials in restenosis prevention using various revascularizationdevices, anti-platelet drugs, anti-thrombotic drugs andanti-inflammatory drugs were uniformly negative (reviewed in Casterellaand Teirstein, Cardiol. Rev. 7: 219-231 [1999]; Andres, Int. J. Mol.Med. 2: 81-89 [1998]; and Rosanio et al., Thromb. Haemost. 82 [suppl 1]:164-170 [1999]). Inspite of all the recent progress, there is still nosatisfactory treatment for stenosis or prevention of restenosis afterballoon angioplasty or stent implantation. Although limited success hasbeen achieved in small randomized trials, stenosis, and particularlyrestenosis, remains a major clinical problem. The instant inventiondiscloses the use of ErbB4 receptor antagonists for the treatment ofstenosis or restenosis by controlling the proliferation of vascularsmooth muscle cells.

The scope of the present invention, however, is not restricted to thedisorders of the vascular smooth muscle cells. The scope specificallyincludes any disorder that results from proliferation of smooth musclecells in any organ and that involves an active role of ErbB4 receptorsand/or corresponding ligands.

Infantile hypertrophic pyloric stenosis (IHPS) is a relatively commondisease that primarily affects young infants. The underlying stenosiscauses functional obstruction of the pyloric canal. Consequently,gastric emptying of milk is disturbed severely. IHPS involveshypertrophy and hyperplasia of the pyloric smooth muscle mass andresults in pyloric stenosis (Oue and Puri, Pediatr. Res. 45: 853-857[1999]). Furthermore, increased expression of EGF, EGF receptor andHB-EGF has been reported in SMCs in pyloric circular and longitudinalmuscle from IHPS patients as compared to control tissues (Shima et al.,Pediatr. Res. 47: 201-207 [2000]). The antagonists of ErbB4 disclosedherein may find use in the control of pyloric smooth muscle cellproliferation and therefore in the treatment of pyloric stenosis.

The contractile nature of smooth muscle cells and regulation of theircontraction by various factors play a crucial role in the urinarycollecting system including bladder, ureters and urethra. Amembrane-bound precursor form of HB-EGF is expressed in urinary bladdersmooth muscle cells and epithelial cells (Freeman et al., J. Clin.Invest. 99: 1028-1036 [1997]; Kaefer et al., J. Urol. 163: 580-584[2000]). Moreover, treatment of bladder SMCs with diphtheria toxin,which is known to utilize membrane-bound HB-EGF as a receptor, inhibitedtheir proliferation (Kaefer et al., ibid). HB-EGF is a potent mitogenfor bladder SMC proliferation, and it acts by binding to ErbB1 (HER1)receptors expressed by these cells, thus acting as an autocrine growthfactor (Borer et al., Lab Invest. 79: 1335-1345 [1999]). The authorsalso demonstrated the expression of ErbB2 and ErbB3 but not ErbB4receptors on bladder SMCs. These findings raise the possibility thatHB-EGF plays a role in the bladder wall thickening that occurs inresponse to obstructive syndromes affecting the lower urinary tract.Therefore, ErbB4 antagonists of the instant invention, particularlyErbB4 immunoadhesin, may prove useful in controlling proliferation ofbladder smooth muscle cells, and consequently in the prevention ortreatment of urinary obstructive syndromes.

The obstructive airway diseases are yet another group of diseases withunderlying pathology involving smooth muscle cell proliferation. Oneexample of this group is asthma which manifests in airway inflammationand bronchoconstriction. EGF has been shown to stimulate proliferationof human airway SMCs and is likely to be one of the factors involved inthe pathological proliferation of airway SMCs in obstructive airwaydiseases (Cerutis et al., Am. J. Physiol. 273: L10-15 [1997]; Cohen etal., Am. J. Respir. Cell. Mol. Biol. 16: 85-90 [1997]). Accordingly, theErbB4 antagonists of the present invention may be used for the treatmentof obstructive airway diseases.

There are two major approaches to introducing the nucleic acid(optionally contained in a vector) into the patient's cells; in vivo andex vivo. For in vivo delivery the nucleic acid is injected directly intothe patient, usually at the site where the chimeric heteroadhesin isrequired. For ex vivo treatment, the patient's cells are removed, thenucleic acid is introduced into these isolated cells and the modifiedcells are administered to the patient either directly or, for example,encapsulated within porous membranes which are implanted into thepatient (see, e.g. U.S. Pat. Nos. 4,892,538 and 5,283,187).

There are a variety of techniques available for introducing nucleicacids into viable cells. The techniques vary depending upon whether thenucleic acid is transferred into cultured cells in vitro, or in vivo inthe cells of the intended host. Techniques suitable for the transfer ofnucleic acid into mammalian cells in vitro include the use of liposomes,electroporation, microinjection, cell fusion, DEAE-dextran, the calciumphosphate precipitation method, etc.

A commonly used vector for ex vivo delivery of the gene is a retrovirus.The currently preferred in vivo nucleic acid transfer techniques includetransfection with viral vectors (such as adenovirus, Herpes simplex Ivirus, or adeno-associated virus) and lipid-based systems (useful lipidsfor lipid-mediated transfer of the gene are DOTMA, DOPE and DC-Chol, forexample). In some situations it is desirable to provide the nucleic acidsource with an agent that targets the target cells, such as an antibodyspecific for a cell surface membrane protein or the target cell, aligand for a receptor on the target cell, etc. Where liposomes areemployed, proteins which bind to a cell surface membrane proteinassociated with endocytosis may be used for targeting and/or tofacilitate uptake, e.g. capsid proteins or fragments thereof tropic fora particular cell type, antibodies for proteins which undergointernalization in cycling, and proteins that target intracellularlocalization and enhance intracellular half-life. The technique ofreceptor-mediated endocytosis is described, for example, by Wu et al.,J. Biol. Chem. 262:4429-4432 (1987); and Wagner et al., Proc. Natl.Acad. Sci. USA 87:3410-3414 (1990). For review of the currently knowngene marking and gene therapy protocols see Anderson et al., Science256:808-813 (1992). See also WO 93/25673 and the references citedtherein.

Therapeutic formulations are prepared for storage by mixing the ErbB4antagonist having the desired degree of purity with optionalphysiologically acceptable carriers, excipients, or stabilizers(Remington's Pharmaceutical Sciences, 16th Edition, Osol., A., Ed.,(1980)), in the form of lyophilized cake or aqueous solutions.Pharmaceutically acceptable carriers, excipients, or stabilizers arenon-toxic to recipients at the dosages and concentrations employed, andinclude buffers such as phosphate, citrate, and other organic acids;antioxidants including ascorbic acid; low molecular weight (less thanabout 10 residues) polypeptides; proteins, such as serum albumin,gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, arginine or lysine; monosaccharides, disaccharides, andother carbohydrates including glucose, mannose, or dextrins; chelatingagents such as EDTA; sugar alcohols such as mannitol or sorbitol;salt-forming counterions such as sodium; and/or nonionic surfactantssuch as Tween™′ Pluronics™, or polyethylene glycol (PEG).

An antibody or an immunoadhesin to be used for in vivo administrationmust be sterile. This is readily accomplished by filtration throughsterile filtration membranes, prior to or following lyophilization andreconstitution. The formulation ordinarily will be stored in lyophilizedform or in solution.

Therapeutic compositions are generally placed into a container having asterile access port, for example, an intravenous solution bag or vialhaving a stopper pierceable by a hypodermic injection needle.

The route of antibody, immunoadhesin or chimeric heteroadhesinadministration is in accord with known methods, e.g., injection orinfusion by intravenous, intraperitoneal, intracerebral, intramuscular,intraocular, intraarterial, or intralesional routes, or bysustained-release systems as noted below. The heteroadhesin or antibodyis administered continuously by infusion or by bolus injection.

Suitable examples of sustained-release preparations includesemipermeable matrices of solid hydrophobic polymers containing theprotein, which matrices are in the form of shaped articles, e.g., films,or microcapsules. Examples of sustained-release matrices includepolyesters, hydrogels (e.g., poly(2-hydroxyethyl-methacrylate) asdescribed by Langer et al., J. Biomed. Mater. Res., 15:167-277 (1981)and Langer, Chem. Tech., 12:98-105 (1982) or poly(vinylalcohol)),polylactides (U.S. Pat. No. 3,773,919, EP 58,481), copolymers ofL-glutamic acid and gamma ethyl-L-glutamate (Sidman et al., Biopolymers,22:547-556 (1983)), non-degradable ethylene-vinyl acetate (Langer etal., supra), degradable lactic acid-glycolic acid copolymers such as theLupron Depot™ (injectable microspheres composed of lactic acid-glycolicacid copolymer and leuprolide acetate), and poly-D-(−)-3-hydroxybutyricacid (EP 133,988).

Sustained-release ErbB4 antagonist also include liposomally entrappeddrug. Liposomes containing ErbB4 antagonist are prepared by methodsknown per se: Epstein et al., Proc. Natl. Acad. Sci. USA 82:3688-3692(1985); Hwang et al., Proc. Natl. Acad. Sci. USA 77:4030-4034 (1980); EP52,322; EP 36,676; EP 88,046; EP 143,949; EP 142,641; Japanese patentapplication 83-118008; U.S. Pat. Nos. 4,485,045 and 4,544,545; and EP102,324. Ordinarily the liposomes are of the small (about 200-800Angstroms) unilamellar type in which the lipid content is greater thanabout 30 mol. % cholesterol, the selected proportion being adjusted forthe optimal therapy. Particularly useful liposomes can be generated bythe reverse phase evaporation method with a lipid composition comprisingphosphatidylcholine, cholesterol and PEG-derivatizedphosphatidylethanolamine (PEG-PE). Liposomes are extruded throughfilters of defined pore size to yield liposomes with the desireddiameter. A chemotherapeutic agent (such as Doxorubicin) is optionallycontained within the liposome. See Gabizon et al. J. National CancerInst. 81(19):1484 (1989).

The ErbB4 antagonist of the invention may be used to bind and sequesterErbB4 ligand or block ErbB4 receptor thereby inhibiting ErbB4 activationin the cell and inhibit cell proliferation. The ErbB4 antagonist of theinvention may be administered to a patient along with other therapy suchas a chemotherapeutic agent. Preparation and dosing schedules for suchchemotherapeutic agents may be used according to manufacturers'instructions or as determined empirically by the skilled practitioner.Preparation and dosing schedules for such chemotherapy are alsodescribed in Chemotherapy Service Ed., M.C. Perry, Williams & Wilkins,Baltimore, Md. (1992). The chemotherapeutic agent may precede, or followadministration of the antagonist or may be given simultaneouslytherewith.

An effective amount of antagonist to be employed therapeutically willdepend, for example, upon the therapeutic objectives, the route ofadministration, and the condition of the patient. Accordingly, it willbe necessary for the therapist to titer the dosage and modify the routeof administration as required to obtain the maximum therapeutic effect.A typical dosage might range from about 1 μg/kg to up to 100 mg/kg ofpatient body weight, preferably about 10 μg/kg to 10 mg/kg. Typically,the clinician will administer antagonist until a dosage is reached thatachieves the desired effect for treatment of the above mentioneddisorders.

5. Methods for Identification of Molecules that Inhibit or Enhance theProliferation or Migration of Smooth Muscle Cells

The present invention discloses a method of screening to identifymolecules that can inhibit or enhance the proliferation of smooth musclecells. For example, a candidate molecule is incubated with a polypeptidecomprising the extracellular domain of an ErbB4 receptor, followed byadding to a culture of smooth muscle cells and determining the effect onthe proliferation of cells. The ErbB4 receptor may be a native ErbB4receptor such as a human ErbB4 receptor, or may be a polypeptide havingat least 85% sequence identity with the amino acid sequence of a nativeErbB4 receptor. The cell proliferation can be monitored and quantitatedin a number of ways. For instance, incorporation of ³H-thymidine intoDNA is a well-established method to monitor cellular DNA synthesisindicative of cell proliferation. The incorporation of ³H-thymidine intoDNA is monitored either microscopically by counting the number of silvergrains in an autoradiograph or biochemically by liquid scintillationcounting. Similarly, incorporation of 5-bromo 2′-deoxyuridine (BrdU)into cellular DNA can be monitored either microscopically orimmunologically. Both assays utilize highly specific monoclonalantibodies that recognize BrdU incorporated into DNA. In the microscopicassay, the cells are permeabilized, reacted with BrdU specificmonoclonal antibodies followed by labeled secondary antibodies. Thesecondary antibodies are detected by virtue of an attached label such asa fluorescent dye (fluorescein isothiocyanate (FITC), rhodamine, TexasRed etc) or an enzymatic label (alkaline phosphatase, horseradishperoxidase etc). A suitable substrate that produces an insoluble productupon enzymatic action is then used to reveal and quantitate the enzymelabeled secondary antibodies. An enzymatic assay monitors the amount ofBrdU specific monoclonal antibodies by a suitable immunoassay such asELISA. The monoclonal antibodies specific for BrdU as well as ELISA kitscontaining such antibodies are available commercially from a number ofsources including Boehringer Mannheim. A flow cytometry can also be usedto monitor cell proliferation. In this method, cells are fractionatedbased on the nuclear DNA content per cell. Since the nuclear DNA contentvaries among cells undergoing division depending on the phase of cellcycle (2n in G1 phase, 4n in G2+M phase and intermediate value in Sphase, wherein n is the value of haploid nuclear DNA content), cellproliferation can be rapidly monitored by estimating the fraction ofcells in S and G2+M phases using this approach.

Since ErbB4-dependent proliferation of smooth muscle cells involvesligand-mediated signal transduction pathway utilizing ErbB4 receptor,any step in this pathway can be monitored and used as a measure of cellproliferation. One such step is a ligand-induced tyrosineautophosphorylation of ErbB4 receptor, which can be monitored by thekinase receptor activation (KIRA) assay as described in WO95/14930. ThisELISA-type assay is suitable for qualitative or quantitative measurementof kinase activation by measuring the autophosphorylation of the kinasedomain of a receptor protein tyrosine kinase such as ErbB4. The firststage of the assay involves phosphorylation of the kinase domain ofErbB4 receptor present in the cell membrane of a smooth muscle cell.Typically, a first solid phase (e.g., a well of a first assay plate) iscoated with a substantially homogeneous population of smooth musclecells. Being adherent cells, the smooth muscle cells adhere naturally tothe first solid phase. One can also use smooth muscle cells transfectedwith a “receptor construct” that comprises a fusion of a kinase receptorand a flag polypeptide. Antibodies specific for flag polypeptide areused in the ELISA part of the assay to capture the receptor with flagpeptide. A candidate molecule and a polypeptide comprising theextracellular domain of a native ErbB4 receptor are then added to thewells containing smooth muscle cells, followed by monitoring tyrosineautophosphorylation of ErbB4 receptor by the KIRA assay. A polypeptidecomprising an amino acid sequence having at least 85% sequence identitywith the amino acid sequence of the extracellular domain of ErbB4receptor can also be used in the assay. Following exposure, the smoothmuscle cells are solubilized using a lysis buffer (which has asolubilizing detergent therein) and gentle agitation, thereby releasingcell lysate which can be subjected to the ELISA part of the assaydirectly, without the need for concentration or clarification of thecell lysate.

The cell lysate thus prepared is then subjected to the second (ELISA)stage of the assay. As a first step in the ELISA stage, a second solidphase (usually a well of an ELISA microtiter plate) is coated with acapture agent (often a capture antibody) which binds specifically toErbB4 receptor or, in the case of a receptor construct, to the flagpolypeptide. Coating of the second solid phase is carried out so thatthe capture agent adheres to the second solid phase. The capture agentis generally a monoclonal antibody but polyclonal antibodies may also beused. The cell lysate obtained is then exposed to, or contacted with,the adhering capture agent so that the receptor or receptor constructadheres to (or is captured in) the second solid phase. A washing step isthen carried out, so as to remove unbound cell lysate, leaving thecaptured receptor or receptor construct. The adhering or capturedreceptor or receptor construct is then exposed to, or contacted with, ananti-phosphotyrosine antibody which identifies phosphorylated tyrosineresidues in the tyrosine kinase receptor. In the preferred embodiment,the anti-phosphotyrosine antibody is conjugated (directly or indirectly)to an enzyme which catalyses a color change of a non-radioactive colorreagent. Accordingly, phosphorylation of the receptor can be measured bya subsequent color change of the reagent. The enzyme can be bound to theanti-phosphotyrosine antibody directly, or a conjugating molecule (e.g,biotin) can be conjugated to the anti-phosphotyrosine antibody and theenzyme can be subsequently bound to the anti-phosphotyrosine antibodyvia the conjugating molecule. Finally, binding of theanti-phosphotyrosine antibody to the captured receptor or receptorconstruct is measured, e.g, by a color change in the color reagent.Anti-phosphotyrosine antibodies that are commercially available can beused for the assay.

The instant invention also provides for a method for screening ofmolecules which can inhibit or enhance migration of smooth muscle cells.One of the formats utilizes a compartmentalized chemotaxis cell culturechambers such as Neuroprobe ChemoTX chemotaxis chambers available from(Neuroprobe Inc., Gaithersburg, Md.). In this method, a porous filterseparates smooth muscle cells in the upper chamber from a mediumcontaining a chemoattractant (e.g. thrombin) in the lower chamber.Smooth muscle cells are incubated with a candidate molecule and apolypeptide comprising the extracellular domain of an ErbB4 receptor. Atthe end of incubation period, the filters are stained and smooth musclecells that have migrated to the bottom of the filter are counted usingan inverted microscope.

A conventional library or a combinatorial library of chemical compoundscan be used for screening purpose. An automated approach adapted forhigh throughput can be conveniently used for the assay. However, thescreening assays are not restricted only to small molecules, evenmacromolecules such as antibodies can be used for the screening.

EXAMPLES

The following examples are offered by way of illustration and not by wayof limitation. The examples are provided so as to provide those ofordinary skill in the art with a complete disclosure and description ofhow to make and use the compounds, compositions, and methods of theinvention and are not intended to limit the scope of what the inventorsregard as their invention. Efforts have been made to insure accuracywith respect to numbers used (e.g. amounts, temperature, etc.) but someexperimental errors and deviation should be accounted for. Unlessindicated otherwise, parts are parts by weight, temperature is indegrees C., and pressure is at or near atmospheric. The disclosures ofall citations in the specification are expressly incorporated herein byreference.

Example 1 Construction, Isolation and Biochemical Characterization ofImmunoadhesins and Chimeric Heteromultimer Immunoadhesins

A unique Mlu I site was engineered into a plasmid expressing human IgGheavy chain (pDR, a gift from J. Ridgeway and P. Carter, Genentech,Inc.) at the region encoding the hinge domain of the immunoglobulin. MluI sites were also engineered into a set of ErbB4 expression plasmids atthe region encoding the ECD/TM junctions of these receptors. Allmutagenesis were performed using the Kunkel method (Kunkel, T., Proc.Natl. Acad. Sci. U.S.A. 82:488 (1985)). The Mlu I sites were utilized tomake the appropriate ErbB4-IgG fusion constructs. The fusion junction ofthe ErbB-IgG chimera was: G⁶⁴⁰ _(ErbB4)-(TR)-DKTH²²⁴ _(VH) where theamino acid numbering of the ErbB4 polypeptide is described in Plowman etal. (Plowman, G. D. et al., (1993a) PNAS USA 90:1746-1750). Theconserved TR sequence is derived from the Mlu I site. The sequence ofthe Fc region used in the preparation of the fusion constructs is foundin Ellison, J. W. et al. (Ellison, J. W. et al. (1982) NAR10:4071-4079). The final expression constructs were in a pRK-typeplasmid backbone wherein eukaryotic expression is driven by a CMVpromoter (Gorman et al., DNA Prot. Eng. Tech. 2:3-10 (1990)).

To obtain protein for in vitro experiments, adherent HEK-293 cells (ATCCNo. CRL-1573) were transfected with the expression plasmid usingstandard calcium phosphate methods (Gorman et al., supra and Huang etal., Nucleic Acids Res. 18:937-947 (1990)). Serum-containing media wasreplaced with serum-free media 15 hours post-transfection and thetransfected cells incubated for 5-7 days. The resulting conditionedmedia was harvested and passed through Protein A columns (1 mL PharmaciaHiTrap™). Purified IgG fusions were eluted with 0.1 M citric acid (pH4.2) into tubes containing 1 M Tris pH 9.0. The eluted proteins weresubsequently dialyzed against PBS and concentrated using Centri-prep-30filters (Amicon). Glycerol was added to a final concentration of 25% andthe material stored at −20° C. Concentrations of material weredetermined via a Fc-ELISA

¹²⁵I-HRG Binding Assay

The EGF-like domain of HRGβ1₍₁₇₇₋₂₄₄₎ was expressed in E. coli, purifiedand radioiodinated as described previously (Sliwkowski, M. et al. J.Biol. Chem. 269:14661-14665 (1994)). Full-length rHRGβ1, which wasexpressed in Chinese hamster ovary cells, was used in Western blotanalysis. Binding assays were performed in Nunc breakapart immuno-moduleplates. Plate wells were coated at 4° C. overnight with 100 μl of 5μg/ml goat-anti-human antibody (Boehringer Mannheim) in 50 mM carbonatebuffer (pH 9.6). Plates were rinsed twice with 200 μl wash buffer(PBS/0.05% Tween-20™) followed by a brief incubation with 100 μl 1%BSA/PBS for 30 min at room temperature. Buffer was removed and each wellwas incubated with 100 μl IgG fusion protein in 1% BSA/PBS undervigorous side-to-side rotation for 1 hour. Plates were rinsed threetimes with wash buffer and competitive binding was carried out by addingvarious amounts of cold competitor γ-HRG and ¹²⁵I-HRGβ1 and incubatingat room temperature for 2-3 hours with vigorous side-to-side rotation.Wells were quickly rinsed three times with wash buffer, drained andindividual wells were counted using a 100 Series Iso Data γ-counter.Scatchard analysis was performed using a modified Ligand program(Munson, P. and Robard, D. (1980) Analytical Biochemistry 107:220-239).

³H-Thymidine Incorporation Assay

Tritiated thymidine incorporation assays were performed in a 96-wellformat. MCF7 cells were plated at 10,000 cells/well in 50:50 F12/DMEM(high glucose) 0.1% fetal calf serum (100 ml). Cells were allowed tosettle for 3 hours, after which ErbB4-IgG fusion proteins and/orheregulin were added to the wells (final volume of 200 ml) and theplates incubated for 15 hours in a 37° C. tissue culture incubator.Tritiated thymidine was added to the wells (20 ml of 1/20 dilutedtritiated thymidine stock: Amersham TRA 120 B363, 1 mCi/ml) and theplates incubated a further 3 hours. Tritiated material was thenharvested onto GF/C unifilters (96 well format) using a PackardFiltermate 196 harvester. Filters were counted using a Packard Topcountapparatus.

Example 2 Effect of ErbB4-IgG Immunoadhesin on Human Aortic SmoothMuscle Cell Proliferation

Human aortic smooth muscle cells (Clonetics) were seeded at about 50%confluent density (5000 cells/well) in 96 well tissue culture plates andincubated overnight in SM2 media (Clonetics). Next day, the media waschanged to M199 supplemented with ITS (1×), 2 mM L-glutamine, 50 μg/mlascorbic acid, 26.5 mM NaHCO3, 100 U/ml penicillin, 100 U/mlstreptomycin and 0.1% (v/v) fetal bovine serum. The cells were furtherincubated for 16 h. The cells were then treated with either Her-4-IgG(400 nM) or buffer for 1 h, followed by treatment with PDGF (100 ng/ml)for 40 h. Control cells were left untreated to estimate the basal levelof cell growth. An aliquot of BrdU (10 μl/well of a 10 μM solution of5-bromo 2′-deoxyuridine prepared in PBS) was added and the cells wereincubated for an additional 2 h. Cell proliferation was monitored byquantitating BrdU incorporation using BrdU ELISA (Cell proliferationkit, Boehringer mannheim, Catalog No 1 647 229) following manufacturer'sinstructions for adherent cells.

As shown in FIG. 5, PDGF stimulated growth of aortic smooth muscle cellsin agreement with earlier reports (Ross et al., Philos. Trans. R. Soc.Lond. B Biol. Sci. 12: 155-169 [1990]). Pre-treatment of cells withErbB4-IgG immunoadhesin reduced the extent of PDGF-stimulatedproliferation of cells. Control cells treated with buffer in place ofErbB4-IgG did not show any significant effect on cell proliferation.These data indicate that at least part of the mitotic response of smoothmuscle cells is mediated by the activation of the ErbB4 receptor, andremoval of ligands which would activate the ErbB4 receptor with theErbB4 immunoadhesin reduces smooth muscle cell proliferation in responseto PDGF.

Example 3 Effect of ErbB4-IgG Immunoadhesin on Human Aortic SmoothMuscle Cell Migration

Human aortic smooth muscle cells were trypsinized and resuspended at aconcentration of 5×10⁵ cells per ml in DME containing 10% FBS. Cellswere pre-incubated with Her-4-IgG (400 nM) or buffer for 15 min. Thelower wells of ChemoTX chemotaxis chambers (Neuroprobe Inc., Cat 116-8)were filled with 300 μl of a solution of 2 U/ml human thrombin or buffer(PBS) negative control. A filter was mounted on top of the chamber andthe smooth muscle cells (buffer or ErbB4 treated) were added to the topwells in a volume of 50 μl. The plate and filter were covered with theclear plastic lid and incubated for 3 h at 37° C. in humidified air with5% CO₂. At the end of the incubation, filters were removed and the topsides were wiped with a Q-tip to remove any remaining cells. The filterswere stained with Dif-Quick staining solution and the number of cellsmigrated to the bottom of the filter were counted using an invertedphase microscope. Six wells in each group and 40 fields in each wellwere counted.

As shown in FIG. 6, thrombin acted as a chemotactic stimulus and inducedmigration of aortic smooth muscle cells. ErbB4-IgG immunoadhesininhibited thrombin-stimulated cell migration. These data indicate thatat least part of thrombin's ability to stimulate smooth muscle cellmigration is mediated by the release of ligand(s) for the ErbB4receptor, and that the removal of these ligands with the ErbB4immunoadhesin reduces the chemotactic response to thrombin

Example 4 Production and Characterization of Anti-ErbB4 MonoclonalAntibodies Generation of Anti-ErbB4 Mabs

A panel of 34 murine monoclonal antibodies which specifically bind theextracellular domain of ErbB4 were produced using conventional hybridomatechnology (Table 2). Total cellular RNA was extracted from MDA-MB-453cells and used as a template in RT PCR to generate the human ErbB4extracellular domain (ECD) coding sequence. Specific oligonucleotidesused in the RT PCR reactions were synthesized on the basis of the ErbB4DNA sequence. A gDErbB4 ECD fusion protein was constructed by ligatingthe coding sequences for amino acids 1-52 of herpes simplex virus type 1glycoprotein D to the sequences encoding amino acids 26-640 of humanErbB4. The gDErbB4 ECD cDNA was inserted into the cytomegalovirus-basedexpression vector pRK5. This construct was transiently transfected intohuman embryonic kidney 293 cells using a standard calcium phosphateprecipitation protocol.

An affinity column was prepared by coupling the anti-gD monoclonal 5B6to CNBR sepharose (Pharmacia LKB Biotechnology, Uppsala Sweden).Supernatant from gDErbB4 ECD transfected 293 cells was concentrated20-40 fold on a ym30 membrane (Amicon, Beverly Mass.) and loaded ontothe affinity resin. The column was washed with PBS and the receptor waseluted with 100 mM acetic acid/500 mM NaCl pH 2.4. The ErbB4 ECD wasbuffer exchanged into PBS and concentrated. Protein concentration wasdetermined by OD280.

Balb/c mice were immunized with approximately 5 μg of ErbB4 ECD in RIBIMPL+TDM+CWS Emulsion (RIBI ImmunoChem Research Inc., Hamilton, Mont.) intheir rear footpads on weeks 0, 1, 2 and 3. The immunized mice weretested for an antibody response by ELISA. The mice with the highesttiters were given an additional 5 μg of ErbB4 ECD in RIBI during week 4.Three days later, the lymphocytes from the popliteal and inguinal nodeswere fused with mouse myeloma line X63-Ag8.653. Fused cells were platedat a density of 200,000 cells per well in 96-well tissue culture platesand hybridoma selection using HAT media supplement (Sigma, St. Louis,Mo.) began one day post fusion. Beginning on day 10, the hybridomasupernatants were screened for the presence of ErbB4 specific antibodiesusing a radioactive capture assay as described below. Stable antibodyproducing clones were obtained by limiting dilution and large quantitiesof specific Mabs were produced in ascites. The antibodies were purifiedon protein A-Sepharose columns (Fermentech, Inc., Edinburgh, Scotland)and stored sterile in PBS at 4° C.

In the radioactive capture assay, Maxisorp breakapart modules (Nunc,Roskilde, Denmark) were coated with 100 μl of 2 μg/ml goat anti-mouseIgG (Boehringer Mannheim) overnight at 4° C. The plates were washed withPBS/0.5% Tween 20 (PBST), blocked with ELISA diluent (PBS/0.5% BSA/0.05%Tween 20) and incubated with monoclonal supernatants for 2 hr at ambienttemperature. The plates were washed and incubated for an additional hourwith 40,000 counts/well of [¹²⁵I]ErbB4 ECD. After washing, the amount ofErbB4 bound to the antibodies was determined by counting the wells on aWallac 1277 GammaMaster (Wallac Inc, Gaithersburg, Md.).

The 34 anti-ErbB4 monoclonal antibodies produced by this method (Table2) have a high affinity for the receptor, exhibit a diversity ofisotypes and are directed to 18 distinct epitopes on the ErbB4 ECD.Isotypes of the antibodies were determined using a Mouse MonoAb ID/SPisotyping kit from Zymed (So. San Francisco, Calif.), followingsupplier's instructions.

Testing the Specificity of Anti-ErbB4 Antibodies

The specificity of the Mabs was determined in an ELISA measuring theirability to bind immobilized HER2, HER3 and ErbB4 extracellular domains(amino acids 1-645, 1-617 and 1-640 respectively). ECDs were coated onELISA plates at a concentration of 1 μg/ml and incubated withbiotinylated anti-ErbB4 Mabs. Bound Mabs were detected usingstreptavidin peroxidase (Sigma, St. Louis, Mo.) and the substrate OPD(Sigma, St. Louis, Mo.). As can be seen in Table 2, nearly all of theantibodies produced were highly specific for ErbB4 (indicated by a ‘4’in the column labeled ‘Specificity’). Four of the antibodies showed somebinding to HER3 (indicated by a ‘3’ in the column labeled‘Specificity’).

Epitope Mapping and Characterization

The ErbB4 epitope bound by each of the monoclonal antibodies wasdetermined by competitive binding analysis (Fendly et al. CancerResearch 50:1550-1558 (1990)). The anti-ErbB4 Mabs were diluted to aconcentration of 25 μg/ml in ELISA diluent and 50 μl was added to anELISA plate precoated with gDErbB4 ECD as above. The plates wereincubated at room temperature for 2 hours and washed with PBST.Dilutions of biotinylated anti-ErbB4 antibodies ranging from 1:1,000 to1:10,000 were prepared and 50 μl was added to the assay plate. Followinga one-hour room temperature incubation, the plates were washed and 50 μlof a 1:5000 dilution of streptavidin peroxidase (Sigma) was added. Theplates were developed using OPD (Sigma). The anti-ErbB4 Mabs weregrouped into epitopes based on their ability to block binding of theothers by 50% or greater in comparison to an irrelevant Mab control. Therelative epitope mapping identified 17 distinct epitopes, identified inTable 2 as A-Q.

The activities of nine representative antibodies were investigatedfurther.

TABLE 2 Summary table of anti-ErbB4 monoclonals Mab Isotype EpitopeSpecificity 4-1440 IgG2b, κ B 4 4-1441 IgG1, κ J 4 4-1459 IgG2a, κ D 44-1460 IgG1, κ C 4 4-1461 IgG2a, κ E 4 4-1462 IgG1, κ C 4 4-1463 IgG2a,κ D 4 4-1464 IgG2b, κ C 4 4-1465 IgG2a, κ L 3, 4 4-1472 IgG2a, κ M 44-1473 IgG2a, κ F 4 4-1474 IgG2b, κ G 4 4-1475 IgG2b, κ P 4 4-1476IgG2a, κ K 4 4-1477 IgG2a, κ Q 4 4-1478 IgG2a, κ I 4 4-1479 IgG2a, κ D 44-1481 IgG2a, κ H 3, 4 4-1482 IgG2b, κ H 4 4-1483 IgG1, κ R 3, 4 4-1484IgG1, κ E 4 4-1485 IgG2a, κ F 4 4-1491 IgG2a, κ G 4 4-1492 IgG2b, κ A 44-1493 IgG2B, κ A 4 4-1494 IgG2b, κ B 4 4-1495 IgG2b, κ A 4 4-1496 IgG1,κ A 3, 4 4-1497 IgG1, κ N 4 4-1498 IgG2b, κ E 4 4-1535 IgG2b, κ B 44-1536 IgG2b, κ A 4 4-1537 IgG2b, κ B 4 4-1543 IgG2a, κ O 4

Determination of Binding Affinity

The relative affinities of the anti-ErbB4 Mabs were determined accordingto the method described by Friguet et al. (J Immunol Methods.77(2):305-19 (1985)). Various concentrations of the ErbB4 ECD (1.1×10⁻⁷M to 1.08×10⁻¹⁰ M) were mixed with a constant concentration ofanti-ErbB4 Mab (2.08×10⁻¹⁰ M) and incubated overnight at 4° C. Followingincubation, the unbound Mabs were assayed by adding 100 μl of thereaction mixture in duplicate to microtiter plates (Nunc) previouslycoated with gDErbB4 ECD (100 μL/well at a concentration of 1 μg/ml in0.05M carbonate buffer, pH 9.6 for 16 hr at 4° C.) and incubated for 1hour at room temperature. After washing with PBST, the bound Mabs weredetected by adding 100 μl/well of a 1:5000 dilution of goat anti-mouseF(ab′)₂ peroxidase (Boehringer Mannheim) for one hour at roomtemperature. The plates were developed using o-phenylenediaminedihydrochloride substrate (OPD, Sigma, St. Louis, Mo.) and read on aplatereader.

The Mabs all showed high affinity binding, with Kd's ranging from 0.4 to12 nm as presented in Table 3.

Non-Reducing Immunoblot

The ability of the anti-ErbB4 Mabs to bind reduced and non-reduced ErbB4ECD was tested by immunoblot analysis. ErbB4 ECD was added to tricinesample buffer, with and without BME, and applied to a 10-20% Novextricine gel (Novex, San Diego, Calif.). The gel was run at 100V andelectroblotted for 60 min. at 0.5 amp onto a PVDF, Immobilon P, membrane(Millipore, Bedford Mass.). The membrane was washed with PBST andblocked overnight with PBS/0.5% BSA/0.1% Tween 20, and incubated with 1μg/ml monoclonal antibody for 1.5 hour at ambient temperature. Themembrane was washed and incubated for an additional hour with a 1:10,000dilution of rat anti-mouse IgG peroxidase (Boehringer Mannheim). Themembrane was washed thoroughly and developed using the Amersham ECLchemiluminescence system (Amersham Life Science Inc., Arlington Heights,Ill.).

None of the Mabs were able to recognize reduced ErbB4 ECD (data notshown), suggesting that they are directed to conformational epitopes.Mabs identified as positive in Table 3 are those that are able torecognize low concentrations of non-reduced ErbB4 ECD. Mabs 4-1459,4-1460, 4-1461, 4-1462, 4-1492 and 4-1497 demonstrated a high level ofimmunoreactivity and were able to bind non-reduced ErbB4 ECD at levelsdown to 0.3 ng.

Inhibition of HRG Binding

A K562 cell line that does not express any EGFR-like receptors was usedto further characterize the anti-ErbB4 monoclonal antibodies. A K562cell line transfected with ErbB4 (1E10.1H4) was produced and cultured inRPMI 1640 with 2 mM L-glutamine (GIBCO/BRL), 10% FBS (Hyclone) and 800μg/ml Geneticin, G418 (Gibco/BRL). At least 20 hr prior to assay,1E10.1H4 was stimulated with 10 nm phorbol-12-myristate, 13-acetate(PMA, Calbiochem, La Jolla Calif.). The anti-ErbB4 Mabs were evaluatedfor their ability to block the binding of HRG to this cell line.

Quadruplicate samples containing 1.0×10⁵ K562 ErbB4 cells resuspended in200 μl of RPMI 1640 with 10 mM HEPES and 0.1% BSA (binding buffer) wereincubated with 132 pM [¹²⁵I]HRGβ1₍₁₇₇₋₂₄₄₎, in the presence of 100 nManti-ErbB4 Mabs, overnight on ice. Following incubation, the cells werecollected using a Multiscreen filtration device (Millipore), and washedtwice with 200 μl ice cold binding buffer. Cell associated counts weremeasured on a gamma counter. The percent binding was calculated againsta control sample containing no Mab. The nonspecific binding wasdetermined by incubation of a sample in the presence of 500 nM coldHRGβ1₍₁₇₇₋₂₄₄₎. Mabs were considered positive for HRG blocking if theyblocked 90% or greater binding. As can be seen in Table 3, six of thenine anti-ErbB4 antibodies tested were able to inhibit ¹²⁵I-HRG bindingat this level. Mab 4-1461 inhibited binding by 7% and 1459 exhibited noHRG blocking. The anti-ErbB4 Mab 4-1497 did not inhibit binding butrather appeared to enhance HRG binding by 26%.

Inhibition of HRG Binding in Human Breast Cancer Cell Lines

Since a number of the anti-ErbB4 Mabs were able to block binding of HRGto transfected K562 cells, their ability to block HRG binding to severalhuman mammary carcinoma cell lines was tested. The cell linesMDA-MB-453, T47D and BT474 (ATCC, Rockville, Md.) were plated into 24well tissue culture plates at a density of 1×10⁵ cells per well andallowed to adhere overnight. The anti-ErbB4 Mabs or anti-HER-2 controlMabs 2C4 and 4D5 were diluted to a concentration of 100 nM in Ham's F-12plus Dulbecco's modified Eagle medium (1:1, v/v) with 10 mM HEPES and0.1% BSA (binding buffer) and added in triplicate to the plates.Following a 30 minute incubation on ice, 1.5×10⁵ counts of [¹²⁵I]HRGβ1₍₁₄₄₋₂₇₇₎ was added. The plates were incubated on ice for fourhours and washed twice with ice cold binding buffer. The cells weresolubilized with 8 M urea/3 M acetic acid and cell associated countswere measured on a Wallac 1277 GammaMaster. The percent binding wascalculated as above. The nonspecific binding was determined byincubation of a sample in the presence of 100 nM cold HRGβ1₍₁₄₄₋₂₇₇₎.

None of the anti-ErbB4 Mabs caused significant inhibition of ¹²⁵IHRGbinding to the carcinoma lines tested. In contrast, the anti-HER-2control Mabs 2C4 and 4D5 blocked binding by 84% and 29% respectively inMDA-MB-453 cells, 70% and 48% in T47D cells and 57% and 12% in BT474cells. The unlabeled HRG control blocked 99% binding in MDA-MB 453cells, 98% binding in T47D cells and 96% binding in BT474 cells at aconcentration of 100 nM. This data suggests that in these cell lines theErbB4 receptor may play a minor role in mediating the HRG responses.

Inhibition of Tyrosine Phosphorylation

Heregulins have been shown to induce the tyrosine phosphorylation ofErbB4. Therefore it was of interest to determine if the anti-ErbB4 Mabswere able to affect HRGβ1₍₁₇₇₋₂₄₄₎ stimulated phosphorylation of thereceptor in the K562 ErbB4 cell line.

The ErbB4 transfected K562 cell line (1E10.H4) was grown in RPMI 1640culture media to a density of 1×10⁶ cells/ml. The cells were thenchanged to serum-free media without PMA (assay buffer) and incubated at37° C. for 2-6 hours. The cells were washed with assay buffer andduplicate samples containing 2.5×10⁵ cells in assay buffer with 0.1%BSA, were incubated with 25 ug of anti-ErbB4 Mabs or a control Mab for30 min. at room temperature. Following incubation, one set of thesamples was stimulated with 15 mM HRGβ1₍₁₇₇₋₂₄₄₎ for 8 minutes at roomtemperature. The supernatants were removed and the cells lysed for 5minutes at 100° C. in 100 μl of SDS sample buffer containing 50 μl/mlβ-mercaptoethanol. A 30 μl aliquot of each sample was electrophoresed ina 4-12% polyacrylamide gel (Novex) and electroblotted onto a PVDFmembrane (Millipore). The membranes were blocked with 2% BSA intris-buffered saline containing 0.05% Tween-20 overnight at 4° C. andincubated with a 1:1000 dilution of recombinant anti-phosphotyrosineperoxidase monoclonal RC20H (Transduction Laboratories, Lexington Ky.)for 4 hours at room temperature. Bound anti-phosphotyrosine Ab wasvisualized using the Amersham ECL system (Amersham Life Science Inc.)and quantified by densitometry.

Six of nine monoclonal antibodies tested inhibited the generation of anHRG-induced tyrosine phosphorylation signal (Table 3). The remainingthree were not inhibitory and none of the anti-ErbB4 Mabs was able tostimulate phosphorylation of the ErbB4 receptor.

Immunohistochemistry

Since anti-ErbB4 Mabs may be useful as diagnostic reagents, theirability to stain frozen cell pellets using standard immunocytochemicaltechniques was investigated. ErbB4 transfected K562 cells (1E10.H4) andthe human breast carcinoma lines MDA-MB-453, T47D, and BT474 (ATCC,Rockville, Md.) were pelleted and frozen in OCT compound (Miles Inc.,Elkhart, Ind.). The frozen pellets were sectioned on a cryostat to athickness of 5 microns, mounted on slides, fixed in cold acetone (4° C.)for 3-5 min. and air-dried. Endogenous peroxidase activity was quenchedusing a modification of the glucose oxidase method. The slides wererinsed with PBS and the cells were blocked for endogenous biotinactivity using a Vector Biotin blocking kit (Vector, Burlingame,Calif.). Endogenous immunoglobulin binding sites were blocked with 10%normal horse serum (Vector). The cells were then incubated with 10 μg/mlanti-ErbB4 Mabs for one hour at RT, followed by a 30 minute incubationwith a 1:200 dilution of biotinylated horse anti-mouse IgG (Vector). Theslides were incubated with ABC Elite Reagent (Vector) for 30 min. andthe ErbB4 receptors visualized using DAB (Pierce, Rockford, Ill.).Mayer's hematoxylin (Rowley Biomedical Institute, Rowley, Mass.) wasused to counterstain the cells.

Many of the anti-ErbB4 Mabs were able to stain the ErbB4 transfectedK562 cells with varying intensity and little or no background staining(Table 3). Numbers represent the intensity of staining compared to anirrelevant control. None of the Mabs was able to stain the frozen humanmammary carcinoma cells that were tested (data not shown).

TABLE 3 Summary table of monoclonal antibody activity Non-Reducing HRGP-Tyr Histo- Mab Isotype Epitope Kd(nM) Immunoblot Blocking Blockingchemistry 4-1440 IgG2b, κ B 1.9 − + + 3+ 4-1459 IgG2a, κ D 0.7 + − − 4+4-1460 IgG1, κ C 1.2 + + + 3+ 4-1461 IgG2a, κ E 2.3 + − − 4+ 4-1462IgG1, κ C 0.4 + + + 2+ 4-1464 IgG2b,κ C 1.0 − + + 2+ 4-1473 IgG2a, κ F6.0 − + + 2-3+ 4-1492 IgG2b, κ A 2.1 + + + − 4-1497 IgG1, κ N 12.0 + − −−

FACS Analysis

To determine whether the anti-ErbB4 Mabs could bind to ErbB4 on thesurface of viable cells, FACS analysis was done using the ErbB4transfected K562 cell line and the mammary carcinoma lines MDA-MB-453,T47D and BT-474. Adherent cells were detached from tissue culture flasksusing 10 mM EDTA in PBS, centrifuged at 1400 rpm for 5 min. andresuspended in PBS with 1% fetal bovine serum (FACS diluent). The cellswere counted, adjusted to 10⁷ cells/ml and 0.1 ml of cells was incubatedwith 10 μg/ml of each Mab in 100 μl FACS diluent for 30 min. at 4° C.The samples were washed, resuspended in 0.1 ml diluent and incubatedwith 1 μg of FITC conjugated F(ab′)₂ fragment of goat anti-mouse IgG(Boehringer Mannheim) for 30 min at 4° C. The cells were washed,resuspended in 0.5 ml FACS diluent and analyzed using a FACScan cellsorter (Becton Dickinson, Mt. View, Calif.). Data was gated by forwardand side scatter and propidium iodide fluorescence to exclude debris,doublets and dead cells.

All of the Mabs bound to the ErbB4 receptor on the ErbB4 transfectedK562 cell line, which is expressed at approximately 2×10⁵receptors/cell. An increase in observed cellular fluorescence of theErbB4 transfected K562 cells from 2 to 50 fold was observed whencompared to the isotype controls. Some of the weaker binding may reflecta ErbB4 ECD epitope that is sequestered on the intact cells. Incontrast, the anti-ErbB4 antibodies 4-1440, 4-1464 and 4-1492, whichgive the highest fluorescence intensity on the transfected cell line,showed minimal binding to the breast carcinoma lines MDA-MB-453, T47Dand BT-474. The positive control anti-HER2 Mab 2-2C4 showed binding tothe tumor lines in proportion to the level of HER-2 expression. Theseresults indicate a level of ErbB4 expression on the MDA-MB-453, T47D andBT-474 cells which is below the detection limit of this assay.

Inhibition of Heregulin Binding to ErbB4 Immunoadhesin

FIG. 7 shows a displacement curve of ¹²⁵IHRG binding to a ErbB4immunoadhesin captured on breakapart modules using the indicatedconcentrations of the anti-ErbB4 Mabs 4-1440, 4-1460, and 4-1464.Maxisorp breakapart modules (Nunc) were coated with 100 μl of a 1:200dilution of goat anti-human Ig (Boehringer Mannheim) in 50 mM carbonatebuffer pH 9.6 overnight at 4° C. The plates were washed with PBST,blocked with ELISA diluent and incubated with 100 μl of 200 ng/ml ErbB4immunoadhesin for 2 hr at ambient temperature. The plates were washedand 50 μl of diluted Mabs (0.1 to 100 nM final) and 50 μl of¹²⁵I-HRGβ1₍₁₇₇₋₂₄₄₎ diluted to give a final concentration of 132 μM wereadded to the plate. Following a 1.5 hr incubation at ambienttemperature, the plates were washed and the amount of ¹²⁵IHRG bound tothe receptor was determined by counting the wells on a Wallac 1277GammaMaster.

FIG. 7 demonstrates that the Mabs inhibited heregulin binding to theimmunoadhesin in a dose dependent manner with ED₅₀ values ranging from0.7 to 1.1 nM. This indicates that the Mabs posses a high degree ofblocking ability.

Deposit of Material

The following hybridomas have been deposited with the American TypeCulture Collection, 10801 University Blvd., Manassas, Va. 20110-2209,USA (ATCC):

Hybridoma ATCC Dep. No. Deposit Date HER4.10H1.1A1 PTA-2828 Dec. 19,2000 HER4.1C6.A11 PTA-2829 Dec. 19, 2000 HER4.3B9.2C9 PTA-2826 Dec. 19,2000 HER4.1A6.5B3 PTA-2827 Dec. 19, 2000 HER4.8B1.2H2 PTA-2825 Dec. 19,2000

Each of the deposited hybridomas produces one of the anti-ErbB4monoclonal antibodies identified in Table 2. HER4.10H1.1A1 produces mAb4-1464, HER4.1C6.A11 produces mAb 4-1440, HER4.3B9.2C9 produces mAb4-1460, HER4.1A6.5B3 produces mAb 4-1492 and HER4.8B1.2H2 produces mAb4-1473

The deposit of the hybridomas with the American Type Culture Collection,Manassas, Va. (ATCC®) was made under the provisions of the BudapestTreaty on the International Recognition of the Deposit of Microorganismsfor the Purpose of Patent Procedure and the Regulations thereunder(Budapest Treaty). This assures maintenance of a viable culture of thedeposit for 30 years from the date of deposit and for the longer of a)at least five (5) years after the most recent request for the furnishingof a sample of the deposit received by the depository, or b) for theenforceable life of a patent issuing from the present application. Thedeposit will be made available by ATTC® under the terms of the BudapestTreaty, and subject to an agreement between Genentech, Inc. andATCC®which assures that all restrictions imposed by the depositor on theavailability to the public of the deposited material will be irrevocablyremoved upon the granting of the pertinent U.S. patent, assurespermanent and unrestricted availability of the progeny of the culture ofthe deposit to the public upon issuance of the pertinent U.S. patent orupon laying open to the public of any U.S. or foreign patentapplication, whichever comes first, and assures availability of theprogeny to one determined by the U.S. Commissioner of Patents andTrademarks to be entitled thereto according to 35 U.S.C. § 122 and theCommissioner's rules pursuant thereto (including 37 C.F.R. § 1.14 withparticular reference to 886 OG 638).

The assignee of the present application has agreed that if a culture ofthe materials on deposit should die or be lost or destroyed whencultivated under suitable conditions, the materials will be promptlyreplaced on notification with another of the same. Availability of thedeposited material is not to be construed as a license to practice theinvention in contravention of the rights granted under the authority ofany government in accordance with its patent laws.

The foregoing written specification is considered sufficient to enableone skilled in the art to practice the invention. The present inventionis not to be limited in scope by the construct deposited, since thedeposited embodiment is intended as a single illustration of certainaspects of the invention and any constructs that are functionallyequivalent are within the scope of this invention. The deposit ofmaterial herein does not constitute an admission that the writtendescription herein contained is inadequate to enable the practice of anyaspect of the invention, including the best mode thereof, nor is it tobe construed as limiting the scope of the claims to the specificillustrations that it represents. Indeed, various modifications of theinvention in addition to those shown and described herein will becomeapparent to those skilled in the art from the foregoing description andfall within the scope of the appended claims.

1. A method for controlling excessive proliferation or migration ofsmooth muscle cells comprising treating said smooth muscle cells with aneffective amount of an antagonist of a native ErbB4 receptor.
 2. Themethod of claim 1 wherein the control is prevention of excessiveproliferation or migration of smooth muscle cells.
 3. The method ofclaim 1 wherein the control is inhibition of excessive proliferation ormigration of smooth muscle cells.
 4. The method of claim 3 wherein saidinhibition is total inhibition.
 5. The method of claim 1 wherein saidsmooth muscle cells are pyloric smooth muscle cells.
 6. The method ofclaim 1 wherein said smooth muscle cells are urinary bladder smoothmuscle cells.
 7. The method of claim 1 wherein said smooth muscle cellsare those of an airway passage.
 8. The method of claim 1 wherein saidexcessive proliferation or migration of smooth muscle cells results instenosis.
 9. The method of claim 1 wherein said smooth muscle cells arevascular smooth muscle cells.
 10. The method of claim 9 wherein saidvascular smooth muscle cells are human.
 11. The method of claim 9wherein said vascular smooth muscle cells are human aortic smooth musclecells.
 12. The method of claim 9 wherein said excessive proliferation ormigration of smooth muscle cells results in vascular stenosis.
 13. Themethod of claim 12 wherein said vascular stenosis is furthercharacterized by excessive proliferation or migration of endothelialcells.
 14. The method of claim 13 wherein said stenosis is restenosis.15. The method of claim 1 wherein the ErbB4 receptor antagonist is animmunoadhesin.
 16. The method of claim 15 wherein said immunoadhesincomprises an extracellular domain sequence of a native ErbB4 receptor.17. The method of claim 16 wherein said native ErbB4 receptor is human.18. The method of claim 17 wherein the native human ErbB4 receptorextracellular domain sequence is fused to an immunoglobulin heavy chainconstant region sequence.
 19. The method of claim 18 wherein saidimmunoglobulin is of IgG isotype.
 20. The method of claim 19 whereinsaid immunoglobulin is of IgG1, IgG2 or IgG3 isotype.
 21. The method ofclaim 19 wherein said immunoadhesin comprises at least one IgGimmunoglobulin light chain.
 22. The method of claim 1 wherein saidantagonist is an antibody.
 23. The method of claim 22 wherein saidantibody is a neutralizing antibody against a native ErbB4 receptor. 24.The method of claim 23 wherein said antibody is a chimeric, humanized orhuman antibody.
 25. The method of claim 23 wherein said antibody isglycosylated.
 26. The method of claim 23 wherein said antibody bindsessentially the same epitope as an antibody produced by a hybridomaselected from the group consisting of HER4.10H1.1A1 (ATCC AccessionNumber PTA-2828), HER4.1C6.A11 (ATCC Accession Number PTA-2829),HER4.3B9.2C9 (ATCC Accession Number PTA-2826), HER4.1A6.5B3 (ATCCAccession Number PTA-2827) and HER4.8B1.2H2 (ATCC Accession NumberPTA-2825).
 27. The method of claim 23 wherein said antibody hascomplementarity determining region (CDR) residues from an antibodyproduced by a hybridoma selected from the group consisting ofHER4.10H1.1A1 (ATCC Accession Number PTA-2828), HER4.1C6.A1 (ATCCAccession Number PTA-2829), HER4.3B9.2C9 (ATCC Accession NumberPTA-2826), HER4.1A6.5B3 (ATCC Accession Number PTA-2827) andHER4.8B1.2H2 (ATCC Accession Number PTA-2825).
 28. A method for treatingstenosis in a mammalian patient comprising administering to said patientan effective amount of an antagonist of a native mammalian ErbB4receptor.
 29. The method of claim 28 wherein said patient is human. 30.The method of claim 29 wherein said stenosis is vascular stenosis. 31.The method of claim 30 wherein said vascular stenosis is restenosis. 32.The method of claim 28 wherein said antagonist is an immunoadhesin. 33.The method of claim 32 wherein said immunoadhesin comprises anextracellular domain sequence of a native human ErbB4 receptor.
 34. Themethod of claim 33 wherein said extracellular domain sequence is fusedto an immunoglobulin heavy chain constant region sequence.
 35. Themethod of claim 34 wherein said immunoglobulin is of IgG isotype. 36.The method of claim 28 wherein said antagonist is an antibody.
 37. Themethod of claim 36 wherein said antibody is a neutralizing antibodyagainst a native human ErbB4 receptor.
 38. The method of claim 36wherein said antibody binds essentially the same epitope as an antibodyproduced by a hybridoma selected from the group consisting ofHER4.10H1.1A1 (ATCC Accession Number PTA-2828), HER4.1C6.A11 (ATCCAccession Number PTA-2829), HER4.3B9.2C9 (ATCC Accession NumberPTA-2826), HER4.1A6.5B3 (ATCC Accession Number PTA-2827) andHER4.8B1.2H2 (ATCC Accession Number PTA-2825).
 39. The method of claim36 wherein said antibody has complementarity determining region (CDR)residues from an antibody produced by a hybridoma selected from thegroup consisting of HER4.10H1.1A1 (ATCC Accession Number PTA-2828),HER4.1C6.A11 (ATCC Accession Number PTA-2829), HER4.3B9.2C9 (ATCCAccession Number PTA-2826), HER4.1A6.5B3 (ATCC Accession NumberPTA-2827) and HER4.8B1.2H2 (ATCC Accession Number PTA-2825).
 40. Themethod of claim 28 wherein said antagonist is administered as aninjection or infusion.
 41. The method of claim 28 wherein said treatmentadditionally reduces hypertension associated with said stenosis.
 42. Themethod of claim 28 wherein said treatment is prevention.
 43. The methodof claim 28 wherein said stenosis is pyloric stenosis.
 44. The method ofclaim 28 wherein said stenosis is thickening of the urinary bladderwall.
 45. The method of claim 28 wherein said stenosis is part of anobstructive airway disease.
 46. A method for treating stenosis in amammalian patient comprising introducing into a cell of said patient anucleic acid encoding an antagonist of an ErbB4 receptor.
 47. The methodof claim 46 wherein said patient is human.
 48. The method of claim 47wherein said antagonist is an immunoadhesin.
 49. The method of claim 48wherein said immunoadhesin comprises an extracellular domain sequence ofa native human ErbB4 receptor fused to an immunoglobulin heavy chainconstant region sequence.
 50. The method of claim 47 wherein saidantagonist is an antibody.
 51. The method of claim 50 wherein saidantibody is a neutralizing antibody against a native ErbB4 receptor. 52.The method of claim 51 wherein said antibody is a chimeric, humanized orhuman antibody.
 53. The method of claim 51 wherein said antibody bindsessentially the same epitope as an antibody produced by a hybridomaselected from the group consisting of HER4.10H1.1A1 (ATCC AccessionNumber PTA-2828), HER4.1C6.A11 (ATCC Accession Number PTA-2829),HER4.3B9.2C9 (ATCC Accession Number PTA-2826), HER4.1A6.5B3 (ATCCAccession Number PTA-2827) and HER4.8B1.2H2 (ATCC Accession NumberPTA-2825).
 54. The method of claim 51 wherein said antibody hascomplementarity determining region (CDR) residues from an antibodyproduced by a hybridoma selected from the group consisting ofHER4.10H1.1A1 (ATCC Accession Number PTA-2828), HER4.1C6.A11 (ATCCAccession Number PTA-2829), HER4.3B9.2C9 (ATCC Accession NumberPTA-2826), HER4.1A6.5B3 (ATCC Accession Number PTA-2827) andHER4.8B1.2H2 (ATCC Accession Number PTA-2825).
 55. The method of claim46 wherein said nucleic acid is introduced in vivo.
 56. The method ofclaim 46 wherein said nucleic acid is introduced ex vivo.
 57. A methodfor treating hypertension associated with vascular stenosis in amammalian patient, comprising administering to said patient an effectiveamount of an antagonist of a native mammalian ErbB4 receptor.
 58. Themethod of claim 57 wherein said antagonist is a small molecule.
 59. Apharmaceutical composition for the treatment of stenosis in a mammalianpatient comprising an effective amount of an antagonist of a nativemammalian ErbB4 receptor, in admixture with a pharmaceuticallyacceptable carrier.
 60. A method for identifying a molecule thatinhibits or enhances the proliferation or migration of smooth musclecells, comprising the steps of: (a) contacting a polypeptide comprisingan amino acid sequence having at least 85% sequence identity with theamino acid sequence of the extracellular domain of a native ErbB4receptor and retaining the ability to control excessive proliferation ormigration of smooth muscle cells, with a candidate molecule; and (b)determining whether the candidate molecule inhibits or enhances theability of said polypeptide to control excessive proliferation ormigration of smooth muscle cells.
 61. The method of claim 60 whereinsaid polypeptide comprises the extracellular domain of a native ErbB4receptor.
 62. The method of claim 61 wherein said receptor is human. 63.The method of claim 61 wherein said polypeptide is an immunoadhesin. 64.The method of claim 60 wherein said molecule enhances the ability ofsaid polypeptide to control excessive proliferation or migration ofsmooth muscle cells.
 65. The method of claim 64 wherein said molecule isselected from the group consisting of antibodies and small molecules.66. An antibody that binds essentially the same epitope of ErbB4 as anantibody produced by a hybridoma selected from the group consisting ofHER4.10H1.1A1 (ATCC Accession Number PTA-2828), HER4.1C6.A11 (ATCCAccession Number PTA-2829), HER4.3B9.2C9 (ATCC Accession NumberPTA-2826), HER4.1A6.5B3 (ATCC Accession Number PTA-2827) andHER4.8B1.2H2 (ATCC Accession Number PTA-2825).
 67. An antibody that hascomplementarity determining region (CDR) residues from an antibodyproduced by a hybridoma selected from the group consisting ofHER4.10H1.1A1 (ATCC Accession Number PTA-2828), HER4.1C6.A11 (ATCCAccession Number PTA-2829), HER4.3B9.2C9 (ATCC Accession NumberPTA-2826), HER4.1A6.5B3 (ATCC Accession Number PTA-2827) andHER4.8B1.2H2 (ATCC Accession Number PTA-2825).
 68. An antibody selectedfrom the group consisting of an antibody produced by a hybridomaselected from the group consisting of HER4.10H1.1A1 (ATCC AccessionNumber PTA-2828), HER4.1C6.A1 (ATCC Accession Number PTA-2829),HER4.3B9.2C9 (ATCC Accession Number PTA-2826), HER4.1A6.5B3 (ATCCAccession Number PTA-2827) and HER4.8B1.2H2 (ATCC Accession NumberPTA-2825).
 69. An antibody that binds essentially the same epitope ofErbB4 bound by an antibody selected from the group consisting ofanti-ErbB4 monoclonal antibodies 4-1440, 4-1460, 4-1473, 4-1492 and4-1464.
 70. An antibody that has complementarity determining region(CDR) residues from an antibody selected from the group consisting ofanti-ErbB4 monoclonal antibodies 4-1440, 4-1460, 4-1473, 4-1492 and4-1464.
 71. An antibody which binds to ErbB4 with high affinity.
 72. Theantibody of claim 71 which binds to ErbB4 with a Kd of less than 100 nM.73. The antibody of claim 71 which binds to ErbB4 with a Kd of less than50 nM.
 74. The antibody of claim 71 which binds to ErbB4 with a Kd ofless than 10 nM.
 75. The antibody of claim 71 which is a humanizedantibody.
 76. The antibody of claim 71 which is a human antibody. 77.The antibody of claim 71 which is an antibody fragment.
 78. An antibodywhich is capable of binding to both ErbB4 and ErbB3.
 79. The antibody ofclaim 78 which binds ErbB4 with high affinity.
 80. The antibody of claim78 which binds both ErbB4 and ErbB3 with high affinity.
 81. An antibodywhich binds to ErbB4 and reduces heregulin binding thereto.
 82. Theantibody of claim 81 which binds ErbB4 with high affinity.
 83. Anantibody which binds to ErbB4 and reduces heregulin-induced tyrosinephosphorylation thereof.
 84. The antibody of claim 83 which binds ErbB4with high affinity.